Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality

ABSTRACT

The present invention relates to methods of improving a treatment outcome comprising administering a heat shock protein (HSP) preparation or an α-2-macroglobulin (α2M) preparation with a non-vaccine treatment modality. In particular, an HSP preparation or an α2M preparation is administered in conjunction with a non-vaccine treatment modality for the treatment of cancer or infectious diseases. In the practice of the invention, a preparation comprising HSPs such as but not limited to, hsp70, hsp90 and gp96 alone or in combination with each other, noncovalently or covalently bound to antigenic molecules or α2M, noncovalently or covalently bound to antigenic molecules is administered in conjunction with a non-vaccine treatment modality.

RELATED APPLICATIONS

This Application claims the benefit under 35 U.S.C. §120 of U.S.application Ser. No. 12/418,724, entitled “USING HEAT SHOCK PROTEINS TOIMPROVE THE THERAPEUTIC BENEFIT OF A NON-VACCINE TREATMENT MODALITY”filed on Apr. 6, 2009, now pending, which is herein incorporated byreference in its entirety. Application Ser. No. 12/418,724 claims thebenefit under 35 U.S.C. §120 of U.S. application Ser. No. 11/283,103,entitled “USING HEAT SHOCK PROTEINS TO IMPROVE THE THERAPEUTIC BENEFITOF A NON-VACCINE TREATMENT MODALITY” filed on Nov. 18, 2005, nowabandoned which is herein incorporated by reference in its entirety.Application Ser. No. 11/283,103 claims the benefit under 35 U.S.C. §120of U.S. application Ser. No. 10/322,312, entitled “USING HEAT SHOCKPROTEINS TO IMPROVE THE THERAPEUTIC BENEFIT OF A NON-VACCINE TREATMENTMODALITY” filed on Dec. 16, 2002, now U.S. Pat. No. 6,984,389, issuedJan. 10, 2006, which is herein incorporated by reference in itsentirety. Application Ser. No. 10/322,312 claims the benefit under 35U.S.C. §120 of U.S. application Ser. No. 10/131,961, entitled “USINGHEAT SHOCK PROTEINS TO IMPROVE THE THERAPEUTIC BENEFIT OF A NON-VACCINETREATMENT MODALITY” filed on Apr. 25, 2002, now abandoned, which isherein incorporated by reference in its entirety.

1. INTRODUCTION

The present invention relates to methods of improving a treatmentoutcome comprising administering a heat shock protein (HSP) preparationor an α-2-macroglobulin (α2M) preparation with a non-vaccine treatmentmodality. In particular, an HSP preparation or an α2M preparation isadministered in conjunction with a non-vaccine treatment modality forthe treatment of cancer or infectious diseases. In the practice of theinvention, a preparation comprising HSPs such as but not limited to,hsp70, hsp90 and gp96 alone or in combination with each other,noncovalently or covalently bound to antigenic molecules or α2M,noncovalently or covalently bound to antigenic molecules is administeredin conjunction with a non-vaccine treatment modality.

2. BACKGROUND OF THE INVENTION

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

2.1. Immune Responses

An organism's immune system reacts with two types of responses topathogens or other harmful agents—humoral response and cell-mediatedresponse (See Alberts, B. et al., 1994, Molecular Biology of the Cell.1195-96). When resting B cells are activated by antigen to proliferateand mature into antibody-secreting cells, they produce and secreteantibodies with a unique antigen-binding site. This antibody-secretingreaction is known as the humoral response. On the other hand, thediverse responses of T cells are collectively called cell-mediatedimmune reactions. There are two main classes of T cells—cytotoxic Tcells and helper T cells. Cytotoxic T cells directly kill cells that areinfected with a virus or some other intracellular microorganism. HelperT cells, by contrast, help stimulate the responses of other cells: theyhelp activate macrophages, dendritic cells and B cells, for example (SeeAlberts, B. et al., 1994, Molecular Biology of the Cell. 1228). Bothcytotoxic T cells and helper T cells recognize antigen in the form ofpeptide fragments that are generated by the degradation of foreignprotein antigens inside the target cell, and both, therefore, depend onmajor histocompatibility complex (MHC) molecules, which bind thesepeptide fragments, carry them to the cell surface, and present themthere to the T cells (See Alberts, B. et al., 1994, Molecular Biology ofthe Cell. 1228). MHC molecules are typically found in abundance onantigen-presenting cells (APCs).

2.3. Chronic Myeloid Leukemia

Chronic myeloid (myelogenous, myelocytic, granulocytic) leukemia (“CML”)is a cancer of the blood and bone marrow characterized by overproductionof white blood cells. CML is characterized by a chronic phase with amedian duration of 3 to 5 years when treated with conventional agentsand an accelerated or acute phase of approximately 3 to 6 monthsduration, inevitably terminating fatally. Initially, the chronic phaseis characterized by no or few symptoms and signs. However, in themajority of cases, constitutional symptoms and abnormal physicalfindings including extramedullary abnormalities, such as myeloblastomas,eventually develop.

CML accounts for 7% to 20% of all leukemias and affects an estimated 1to 2/100,000 persons in the general population. The American CancerSociety estimates that there will be about 4,400 new cases of CML in theUnited States this year.

CML is caused by a specific cytogenetic abnormality, the Philadelphia(“Ph+”) chromosome, which results in a clonal myeloproliferativedisorder of pluripotent hematopoietic stem cells (Faderl et al., 1999,New England 3. Med. 341(3):164-172). The Ph+ chromosome results from abalanced translocation between the long arms of chromosomes 9 and 22,resulting in the bcr/abl chimeric gene that expresses an abnormal fusionprotein with altered tyrosine kinase activity.

Current treatment options for patients in the chronic phase of CMLinclude busulfan (BUS), hydroxyurea (HU), interferon (IFN)-basedregimens, specific kinase inhibitor for bcr/abl or bone marrow/stem celltransplantation (BMT) (Silver et al., 1999, Blood 94(5):1517-1536).Until a few years ago, allogeneic BMT was the treatment of choice forall eligible patients, because it was the only treatment that appearedto change the natural course of the disease. Studies showed that atleast half of the patients transplanted remain alive 5 to 10 years afterthe treatment. However, this practice was still complicated by the lackof donors, and the significant transplant related complications such asgraft versus host diseases and infections. IFN-based regimens have alsoinfluenced the natural course of CML. However, IFN-based regiments aloneonly offer survival advantage by a median of about 20 months (ChronicMyeloid Leukemia Trialists' Collaborative Group, 1997, J. Natl. CancerInst. 89(21):1616-20).

Specific bcr/abl inhibitors such as Gleevec™ (imatinib mesylate,Novartis™) have shown promise in the phase I clinical trials. Druckerand Lydon, 2000, J. Clin. Invest. 105(1):3-7; Dazzi et al., 2000,Leukemia 14:419-426; see also Hellman, Principles of Cancer Management:6th edition, 2001, DeVita et al., eds., J. B. Lippencott Company,Philadelphia, which is hereby incorporated by reference in its entirety,pp. 2443-2444. Gleevec™ (imatinib mesylate) is also known as signaltransduction inhibitor 571, STI-571, and CGP 57148. Based on the phaseII studies (Druker et al., 2001, New England J. Med. 344(14): 1031-1037,and Druker et al., 2001, New England J. Med. 344(14):1038-1042) the FDAhas approved the use of Gleevec™ to treat the following three phases ofCML: chronic phase that is no longer responding to the standard therapy,interferon; accelerated phase; and myeloid blast crisis. The long-termefficacy and toxicity, however, remain unknown. Furthermore, adverseeffects have been observed in Gleevec™-treated patients including edema,hepatotoxicity, and hematologic toxicity. Physician's Desk Reference(56^(th) ed., 2002). In addition, resistance to Gleevec™ has alreadybeen described. Le Coutre et al., 2000, Blood 95(5): 1758-1766. Thus,there is a need in the art for improved methods of treating CML.

2.4. Heat Shock Proteins

Heat shock proteins (HSPs), which are also referred to interchangeablyherein as stress proteins, can be selected from among any cellularprotein that satisfies the following criteria. It is a protein whoseintracellular concentration increases when a cell is exposed to astressful stimuli, it is capable of binding other proteins or peptides,it is capable of releasing the bound proteins or peptides in thepresence of adenosine triphosphate (ATP) or low pH, and it is a proteinshowing at least 35% homology with any cellular protein having any ofthe above properties. HSPs include constitutively expressed conservedcellular homologs of the proteins induced by stress. Therefore it iscontemplated that stress proteins/HSPs include other proteins, muteins,analogs, and variants thereof having at least 35% to 55%, preferably 55%to 75%, and most preferably 75% to 85% amino acid identity with membersof the three families with the above properties.

The first stress proteins to be identified were the HSPs. As their nameimplies, HSPs are synthesized by a cell in response to heat shock. Todate, three major families of HSPs have been identified based onmolecular weight. The families have been called hsp60, hsp70 and hsp90where the numbers reflect the approximate molecular weight of the stressproteins in kilodaltons. Many members of these families were foundsubsequently to be induced in response to other stressful stimuliincluding, but not limited to, nutrient deprivation, metabolicdisruption, oxygen radicals, and infection with intracellular pathogens.(See Welch, May 1993, Scientific American 56-64; Young, 1990, Annu. Rev.Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething, et al.,1992, Nature 355:33-45; and Lindquist, et al., 1988, Annu. Rev. Genetics22:631-677), the disclosures of which are incorporated herein byreference. It is contemplated that lisps/stress proteins belonging toalt of these three families can be used in the practice of the instantinvention.

HSPs are intracellular molecules that are abundant, soluble, and highlyconserved. As intracellular chaperones, HSPs participate in manybiochemical pathways of protein maturation and function active duringtimes of stress and normal cellular homeostasis. Many stresses candisrupt the three-dimensional structure, or folding, of a cell'sproteins. Left uncorrected, mis-folded proteins form aggregates that mayeventually kill the cell. HSPs bind to those damaged proteins, helpingthem refold into their proper conformations. In normal (unstressed)cellular homeostasis, HSPs are required for cellular metabolism. HSPshelp newly synthesized polypeptides fold and thus prevent prematureinteractions with other proteins. Also, HSPs aid in the transport ofproteins throughout the cell's various compartments.

The major HSPs can accumulate to very high levels in stressed cells, butthey occur at low to moderate levels in cells that have not beenstressed. For example, the highly inducible mammalian hsp70 is hardlydetectable at normal temperatures but becomes one of the most activelysynthesized proteins in the cell upon heat shock (Welch et al., 1985, J.Cell. Biol. 101:1198-1211). In contrast, hsp90 and hsp60 proteins areabundant at normal temperatures in most, but not all, mammalian cellsand are further induced by heat (Lai at al., 1984, Mol. Cell. Biol.4:2802-2810; van Bergen en Henegouwen et al., 1987, Genes Dev.1:525-531).

HSPs have been found to have immunological and antigenic properties.Immunization of mice with gp96 or p84/86 isolated from a particulartumor rendered the mice immune to that particular tumor, but not toantigenically distinct tumors (Srivastava, P. K. et al., 1988,Immunogenetics 28:205-207; Srivastava, P. K. et al., 1991, Curr. Top.Microbiol. Immunol. 167:109-123). Further, hsp70 was shown to elicitimmunity to the tumor from which it was isolated but not toantigenically distinct tumors. However, hsp70 depleted of peptides wasfound to lose its specific immunogenic activity (Udono, M., andSrivastava, P. K., 1993, J. Exp. Med. 178:1391-1396). These observationssuggested that the heat shock proteins are not antigenic per se, butform noncovalent complexes with antigenic peptides, and the complexescan elicit specific immunity to the antigenic peptides (Srivastava, P.K., 1993, Adv. Cancer Res. 62:153-177; Udono, H. et al., 1994, J.Immunol., 152:5398-5403; Suto, R. et al., 1995, Science, 269:1585-1588).Recently, hsp60 and hsp70 have been found to stimulate production ofproinflammatory cytokines, such as TNFα and IL-6, by monocytes,macrophages, or cytotoxtic T cells (Breloer et al., 1999, J. Immunol.162:3141-3147; Chen at, 1999, J. Immunol. 162:3212-3219; Ohashi at,2000, J. Immunol. 164:558-561; Mea et at, 2000, Nature Medicine,6:435-442; Todryk et al., 1999, J. Immunol. 163:1398-1408). Hsp70 hasalso been shown to target immature dendritic cells and make them moreable to capture antigens (Todryk et at, J. Immunol. 163:1398-1408). Ithas been postulated that release of or induction of expression of hsp60and hsp70, e.g., due to cell death, may serve to signal that an immunereaction should be raised (Chen at, 1999, J. Immunol. 162:3212-3219;Ohashi et al., 2000, J. Immunol. 164:558-561; Todryk et al., 1999, J.Immunol. 163:1398-1408).

The use of noncovalent complexes of HSP and peptide, purified fromcancer cells, for the treatment and prevention of cancer has beendescribed in U.S. Pat. Nos. 5,750,119, 5,837,251, and 6,017,540.

The use of HSP-peptide complexes for sensitizing antigen presentingcells in vitro for use in adoptive immunotherapy is described in U.S.Pat. Nos. 5,985,270 and 5,830,464.

HSP-peptide complexes can also be isolated from pathogen-infected cellsand used for the treatment and prevention of infection caused by thepathogen, such as viruses, and other intracellular pathogens, includingbacteria, protozoa, fungi and parasites; see U.S. Pat. Nos. 5,961,979,and 6,048,530.

Immunogenic HSP-peptide complexes can also be prepared by in vitrocomplexing of HSPs and antigenic peptides, and the uses of suchcomplexes for the treatment and prevention of cancer and infectiousdiseases has been described in U.S. Pat. Nos. 5,935,576, and 6,030,618.The use of heat shock protein in combination with a defined antigen forthe treatment of cancer and infectious diseases have also been describedin PCT publication WO97/06821 dated Feb. 27, 1997.

The purification of HSP-peptide complexes from cell lysate has beendescribed previously; see for example, U.S. Pat. Nos. 5,750,119, and5,997,873.

2.5. α2-Macroglobulin

The α-macroglobulins are members of a protein superfamily ofstructurally related proteins which also comprises complement componentsC3, C4 and C5. The human plasma protein alpha(2)macroglobulin (α2M) is a720 kDa homotetrameric protein primarily known as proteinase inhibitorand plasma and inflammatory fluid proteinase scavenger molecule (forreview see Chu and Pizzo, 1994, Lab. Invest. 71:792). Alpha (2)macroglobulin is synthesized as a 1474 amino acid precursor, the first23 of which function as a signal sequence that is cleaved to yield a1451 amino acid mature protein (Kan et al., 1985, Proc. Natl. Acad. Sci.U.S.A. 82:2282-2286).

Alpha(2)macroglobulin promiscuously binds to proteins and peptides withnucleophilic amino acid side chains in a covalent manner (Chu et al.,1994, Ann. N.Y. Acad. Sci. 737:291-307) and targets them to cells whichexpress the α2M receptor (α2MR) (Chu and Pizza, 1993, J. Immunol.150:48). Binding of α2M to the α2MR is mediated by the C-terminalportion of α2M (Holtet et al., 1994, FEBS Lett. 344:242-246) and keyresidues have been identified (Nielsen et al., 1996, J. Biol. Chem.271:12909-12912).

Generally known for inhibiting protease activity, α2M binds to a varietyof proteases thorough multiple binding sites (see, e.g., Hall et al.,1981, Biochem. Biophys. Res. Commun. 100(1):8-16). Protease interactionwith α2M results in a complex structural rearrangement calledtransformation, which is the result of a cleavage within the “bait”region of α2M after the proteinase becomes “trapped” by thioesters. Theconformational change exposes residues required for receptor binding,allowing the α2M-proteinase complex to bind to the α2MR. Methylamine caninduce similar conformational changes and cleavage as that induced byproteinases. The uncleaved form of α2M, which is not recognized by thereceptor, is often referred to as the “slow” form (s-α2M). The cleavedform is referred to as the “fast” form (f-α2M) (reviewed by Chu et al.,1994, Ann. N.Y. Acad. Sci. 737:291-307).

Studies have shown that, in addition to its proteinase-inhibitoryfunctions, α2M, when complexed to antigens, can enhance the antigens'ability to be taken up by antigen presenting cells such as macrophagesand presented to T cell hybridomas in vitro by up to two orders ofmagnitude (Chu and Pizzo, 1994, Lab. Invest. 71:792), and induce T cellproliferation (Osada et al., 1987, Biochem. Biophys. Res. Commun.146:26-31). Further evidence suggests that complexing antigen with α2Menhances antibody production by crude spleen cells in vitro (Osada etal., 1988, Biochem. Biophys. Res. Commun. 150:883) and elicits an invivo antibody responses in experimental rabbits (Chu et al., 1994, J.Immunol. 152:1538-1545) and mice (Mitsuda et al., 1993, Biochem.Biophys. Res. Commun. 101:1326-1331). However, none of these studieshave shown whether α2M-antigen complexes are capable of elicitingcytotoxic T cell responses in vivo.

α2M can form complexes with antigens, which are taken up by antigenpresenting cells (“APCs”) via the α2MR, also known as LDL (low-densitylipoprotein) Receptor-Related Protein (“LRP”) or CD91 (seePCT/US01/18047, which is incorporated by reference herein in itsentirety). α2M directly competes for the binding of heat shock proteingp96 to the α2MR, indicating that α2M and hsps may bind to a commonrecognition site on the α2MR (Binder et al., 2000, Nature Immunology1(2), 151-154). Additionally, α2M-antigenic peptide complexes preparedin vitro can be administered to animals to generate a cytotoxic T cellresponse specific to the antigenic molecules (Binder et al., 2001, J.Immunol. 166:4968-72). Thus, because hsps and α2M have a number ofcommon functional attributes, such as the ability to bind peptide, therecognition and uptake by the α2MR, and the stimulation of a cytotoxic Tcell response, α2M can be used for immunotherapy against cancer andinfectious diseases.

3. SUMMARY OF THE INVENTION

The present invention is based, in part, on the recognition that an HSPpreparation can enhance or improve the therapeutic benefit ofnon-vaccine treatment modalities or therapeutic modalities for treatmentof cancer or infectious diseases. Thus, the present inventionencompasses methods and compositions that comprise administering an HSPpreparation in combination with a non-vaccine treatment modality. Alsoencompassed are methods and compositions that comprise administering anα2M preparation in combination with a non-vaccine treatment modality. Inparticular, the invention encompasses methods and compositions oftreatment and compositions that provide a better therapeutic profilethan that of an HSP preparation or α2M preparation administered alone ora non-vaccine treatment modality administered alone. The source of theHSP or α2M is preferably an eukaryote, and most preferably a mammal. Thesubject receiving the treatment is preferably a mammal including, butnot limited to, domestic animals, such as cats and dogs; wild animals,including foxes and raccoons; livestock and fowl, including horses,cattle, sheep, turkeys and chickens, as well as any rodents. Mostpreferably, the subject is human.

The invention provides methods for improving the therapeutic outcome ofa non-vaccine treatment modality comprising administering either an HSPpreparation or an α2M preparation, preferably a purified HSP preparationor a purified α2M preparation, in conjunction with the administration ofthe treatment modality. Either the HSP preparation or the α2Mpreparation can be administered over a period of time which may precede,overlap, and/or follow a treatment regimen with a non-vaccine treatmentmodality. The HSP preparation or the α2M preparation can be administeredconcurrently, before, or after the administration of the treatmentmodality. Examples of treatment modalities include but are not limitedto antibiotics, antivirals, antifungal compounds, anti-cancer treatmentssuch as chemotherapeutic agents, and radiation, as well as biologicaltherapeutic agents and immunotherapeutic agents. In preferredembodiments, the treatment modality is useful in the treatment orprevention of cancer. In particularly preferred embodiments, thetreatment modality is useful in the treatment or prevention of chronicmyelgenous leukemia or soft tissue sarcomas including but not limited togastrointestinal stromal tumors. In another preferred embodiment, thetreatment modality is Gleevec™.

In one embodiment, the invention encompasses methods of treatment thatprovide better therapeutic profiles than the administration of thetreatment modality or the HSP preparation alone. In another embodiment,the invention encompasses methods of treatment that provide bettertherapeutic profiles than the administration of the treatment modalityor the α2M preparation alone. Encompassed by the invention are methodswherein the administration of a treatment modality with an HSPpreparation or an α2M preparation has additive potency or additivetherapeutic effect. The invention also encompasses synergistic outcomeswhere the therapeutic efficacy is greater than additive. Preferably,such administration of a treatment modality with an HSP preparation orwith an α2M preparation also reduces or avoids unwanted or adverseeffects. Given the invention, in certain embodiments, doses ofnon-vaccine treatment modality can be reduced or administered lessfrequently, preferably increasing patient compliance, improving therapyand/or reducing unwanted or adverse effects. In a specific embodiment,lower or less frequent doses of chemotherapy or radiation therapy areadministered to reduce or avoid unwanted effects. Alternatively, dosesof HSP preparation and doses of α2M preparation can be reduced oradministered less frequently if administered with a treatment modality.

In one embodiment, the present invention provides a method for improvingthe outcome of a treatment in a subject receiving a therapeutic modalitywhich is not a vaccine. The method comprises administering either a heatshock protein preparation, preferably a purified HSP preparation, or anα2M preparation, preferably a purified α2M preparation, to the subjectbefore, concurrently with, or after the administration of thetherapeutic modality. In a specific embodiment, the HSP preparation orthe α2M preparation can augment the therapeutic benefit of a treatmentmodality and improve the outcome of the treatment. Without being boundby any theory or mechanism, the administration of a mammalian HSPpreparation or α2M preparation to a subject can enhance theresponsiveness of non-specific immune mechanisms of the subject, forexample, by increasing the number of natural killer (NK) cells and/oraccelerating the maturation of dendritic cells and/or can also enhancethe responsiveness of specific immune mechanisms, such as by increasingthe number of CD4+ and CD8+ T cells. In a preferred specific embodiment,the HSP preparation is administered before the administration of thetherapeutic modality. In another preferred specific embodiment, the α2Mpreparation is administered before the administration of the therapeuticmodality.

In another embodiment, the present invention provides a method forimproving the outcome of a treatment in a subject receiving an HSPpreparation, preferably a purified HSP preparation, by administering anon-vaccine therapeutic modality to the subject before, concurrentlywith, or after the administration of the HSP preparation. In a specificembodiment, the non-vaccine therapeutic modality can augment thetherapeutic benefit of an HSP preparation and improve the outcome of thetreatment.

In another embodiment, the present invention provides a method forimproving the outcome of a treatment in a subject receiving an α2Mpreparation, preferably a purified α2M preparation, by administering anon-vaccine therapeutic modality to the subject before, concurrentlywith, or after the administration of the α2M preparation. In a specificembodiment, the non-vaccine therapeutic modality can augment thetherapeutic benefit of an α2M preparation and improve the outcome of thetreatment.

In certain embodiments, the administration of the HSP/α2M preparation inthe absence of administration of the therapeutic modality or theadministration of the therapeutic modality in the absence ofadministration of the HSP/α2M preparation is not therapeuticallyeffective. In a specific embodiment, the amount of HSP/α2M preparationor therapeutic modality is administered in an amount insufficient to betherapeutically effective alone. In alternate embodiments, both or atleast one of the HSP/α2M preparation or therapeutic modality istherapeutically effective when administered alone.

In various embodiments, the methods comprise the administration of anHSP preparation, preferably a purified HSP preparation, to a subjectreceiving a treatment modality for the treatment of cancer or infectiousdiseases. Preferably the HSP preparation comprises HSP-peptide complexesdisplaying the antigenicity of a tumor specific antigen or tumorassociated antigen of the type of cancer or an antigen of an infectiousagent, i.e., heat shock proteins complexed to antigenic peptides of thecancer cells or infected cells from which the complexes are obtained.Accordingly, in one embodiment, the specific immunogenicity of the HSPpreparation derives from the peptide complexed to the HSP. In preferredembodiments, the HSP-peptide complexes are isolated from an antigensource such as cancer tissues, cancer cells, or infected tissues. In thepractice of the invention, such HSP-peptide complexes are preferably,autologous to the individual subject, i.e., obtained from the tissues ofthe subject receiving the administration of HSP preparation andtreatment modality, but need not be (i.e., allogeneic to the individualsubject).

In various other embodiments, the methods comprise the administration ofan α2M preparation, preferably a purified α2M preparation, to a subjectreceiving a treatment modality for the treatment of cancer or infectiousdiseases. Preferably the α2M preparation comprises α2M-peptide complexesdisplaying the antigenicity of a tumor specific antigen or tumorassociated antigen of the type of cancer or an antigen of an infectiousagent, i.e., α2M complexed to antigenic peptides of the cancer cells orinfected cells from which the complexes are obtained. Accordingly, inone embodiment, the specific immunogenicity of the α2M preparationderives from the peptide complexed to the α2M. In preferred embodiments,the α2M-peptide complexes are isolated from an antigen source such ascancer tissues, cancer cells, or infected tissues. In the practice ofthe invention, such α2M-peptide complexes are preferably, autologous tothe individual subject, i.e., obtained from the tissues of the subjectreceiving the administration of α2M preparation and treatment modality,but need not be (i.e., allogeneic to the individual subject).

In one embodiment, the methods comprise the administration of an HSPpreparation or an α2M preparation, preferably a purified HSP preparationor a purified α2M preparation, to a subject receiving a treatmentmodality for treatment of an infectious disease. Such treatmentmodalities are known in the art and include but are not limited toantibiotics, antivirals, antifungals as well as biological andimmunotherapeutic agents. Preferably the HSP preparation comprisesHSP-peptide complexes which display the antigenicity of an agent of theinfectious disease. Preferably the α2M preparation comprises α2M-peptidecomplexes which display the antigenicity of an agent of the infectiousdisease. In a specific embodiment, the outcome of a treatment of a typeof infectious disease in a subject receiving a non-vaccine therapeuticmodality is improved by administering HSP-peptide complexes comprisingan HSP complexed to a peptide that displays the antigenicity of anantigen of an agent of said type of infectious disease. Preferably, theHSP-peptide complexes are not present in admixture with HSP or α2M thatis not complexed to a peptide that displays the antigenicity of anantigen of an agent of the same infectious disease. (See InternationalApplication No. PCT/US01/28840, filed Sep. 15, 2001, incorporated byreference herein in its entirety). In one embodiment, the HSPpreparation is administered prior to administration of the therapeuticmodality. In another embodiment, the therapeutic modality isadministered prior to the administration of the HSP preparation. Inanother specific embodiment, the outcome of a treatment of a type ofinfectious disease in a subject receiving a non-vaccine therapeuticmodality is improved by administering α2M-peptide complexes comprisingan α2M complexed to a peptide that displays the antigenicity of anantigen of an agent of said type of infectious disease. Preferably, theα2M-peptide complexes are not present in admixture with HSP or α2M thatis not complexed to a peptide that displays the antigenicity of anantigen of an agent of the same infectious disease. In one embodiment,the α2M preparation is administered prior to administration of thetherapeutic modality. In another embodiment the therapeutic modality isadministered prior to the administration of the α2M preparation.

In another embodiment, the methods comprise the administration of eitheran HSP preparation or an α2M preparation, preferably a purified HSPpreparation or a purified α2M preparation, to a subject receiving atreatment modality for treatment of cancer. Such treatment modalitiesinclude but are not limited to chemotherapies and radiation therapies aswell as hormonal therapies, biological therapies and immunotherapies.Preferably the HSP preparation or α2M preparation is administered to asubject receiving chemotherapy or radiation therapy for treatment ofcancer. Preferably the HSP preparation comprises HSP-peptide complexeswhich display the antigenicity of the type of cancer being treated.Preferably where the preparation is an α2M preparation, the α2Mpreparation comprises α2M-peptide complexes which display theantigenicity of the type of cancer being treated. Accordingly, inpreferred embodiments, the invention provides methods for improving theoutcome of cancer treatment in a subject receiving a therapeuticmodality which is not a vaccine using HSP-peptide complexes comprisingan HSP complexed to a peptide that displays the antigenicity of a tumorspecific antigen or tumor associated antigen of a type of cancer orusing α2M-peptide complexes comprising an α2M complexed to a peptidethat displays the antigenicity of a tumor specific antigen or tumorassociated antigen of a type of cancer. In certain preferredembodiments, such HSP-peptide complexes and α2M-peptide complexes arenot diluted with either HSP or α2M that is not complexed to a peptidethat displays the antigenicity of an antigen of the same type of cancer.In one embodiment, the HSP preparation or α2M preparation isadministered prior to administration of the therapeutic modality. Inanother embodiment, the therapeutic modality is administered prior toadministration of the HSP preparation or α2M preparation.

In various embodiments, the HSP preparation or α2M preparation isadministered with an anti-cancer agent which can be but is not limitedto a cytotoxic agent, antimitotic agent, tubulin stabilizing agent,microtubule formation inhibiting agent, topoisomerase inhibitors,alkylating agent, DNA interactive agent, antimetabolite, RNA/DNAantimetabolite, DNA antimetabolite. In a specific embodiment, theanti-cancer agent is a chemotherapeutic.

In a specific embodiment, an HSP preparation is administered to asubject receiving a chemotherapeutic agent for treatment of cancer. Inanother preferred embodiment, an α2M preparation is administered to asubject receiving a chemotherapeutic agent for treatment of cancer. Suchchemotherapeutic agents are known in the art and include but are notlimited to: methotrexate, taxol, mercaptopurine, thioguanine,hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas,cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine,etoposides, campathecins, bleomycin, doxorubicin, idarubicin,daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase,vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel,doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel andpaclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide,nitrosoureas such as carmustine and lomustine, vinca alkaloids, platinumcompounds, mitomycin, gemcitabine, hexamethylmelamine, topotecan,tyrosine kinase inhibitors, tyrphostins, STI-571 or Gleevec™ (imatinibmesylate), herbimycin A, genistein, erbstatin, and lavendustin A.

In preferred embodiments, each of the methods above compriseadministering either an HSP preparation or an α2M preparation,preferably a purified HSP preparation or a purified α2M preparation, toa subject receiving a drug of the 2-phenylaminopyrimidine class fortreatment of cancer. More preferably, the subject is receiving Gleevec™(i.e., imatinib mesylate) for treatment of cancer.

In another specific embodiment, an HSP preparation or an α2M preparationis administered to a subject receiving radiation therapy for treatmentof cancer. For radiation treatment, the radiation can be gamma rays orX-rays. The methods encompass treatment of cancer comprising radiationtherapy, such as external-beam radiation therapy, interstitialimplantation of radioisotopes (I-125, palladium, iridium), radioisotopessuch as strontium-89, thoracic radiation therapy, intraperitoneal P-32radiation therapy, and/or total abdominal and pelvic radiation therapy.For a general overview of radiation therapy, see Hellman, Chapter 16:Principles of Cancer Management: Radiation Therapy, 6th edition, 2001,DeVita et al., eds., J.B. Lippencott Company, Philadelphia. In preferredembodiments, the radiation treatment is administered as external beamradiation or teletherapy wherein the radiation is directed from a remotesource. In various preferred embodiments, the radiation treatment isadministered as internal therapy or brachytherapy wherein a radioactivesource is placed inside the body close to cancer cells or a tumor mass.

In another embodiment, each of the above methods comprise theadministration of HSP preparation, preferably a purified HSPpreparation, to a subject receiving a combination of treatmentmodalities for the treatment of cancer. In another embodiment, each ofthe above methods comprise the administration of an α2M preparation,preferably a purified α2M preparation, to a subject receiving acombination of treatment modalities for the treatment of cancer.Preferably the HSP preparation and α2M preparation each comprisesHSP-peptide complexes and α2M-peptide complexes, respectively, whichdisplay the antigenicity of the type of cancer being treated. In onesuch embodiment, an HSP preparation is administered to a subjectreceiving chemotherapy in combination with a biological therapy,preferably a cytokine. In another such embodiment, an α2M preparation isadministered to a subject receiving chemotherapy in combination with abiological therapy, preferably a cytokine. In various embodiments, thecytokine is selected from the group consisting of IL-1α, IL-1β, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFNα,IFNβ, IFNγ, TNFα, TNFβ, G-CSF, GM-CSF, TGF-β, IL-15, IL-18, GM-CSF,INF-γ, INF-α, SLC, endothelial monocyte activating protein-2 (EMAP2),MIP-3α, MIP-3β, or an MHC gene, such as HLA-B7. Additionally, otherexemplary cytokines include other members of the TNF family, includingbut not limited to TNF-α-related apoptosis-inducing ligand (TRAIL),TNF-α-related activation-induced cytokine (TRANCE), TNF-α-related weakinducer of apoptosis (TWEAK), CD40 ligand (CD40L), LT-α, LT-β, OX4OL,CD4OL, FasL, CD27L, CD30L, 4-1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17,and AITR-L, or a functional portion thereof. See, e.g., Kwon et at.,1999, Curr. Opin. Immunol. 11:340-345 for a general review of the TNFfamily. In one embodiment, the HSP preparation is administered prior tothe treatment modalities. In another embodiment, the treatment modalityis administered prior to the HSP preparation.

In a preferred embodiment, a purified HSP preparation is administered toa subject receiving cyclophosphamide in combination with IL-12 fortreatment of cancer. In another preferred embodiment, a purified α2Mpreparation is administered to a subject receiving cyclophosphamide incombination with IL-12 for treatment of cancer.

In another embodiment, the above methods are useful for the preventionof cancer or infectious diseases. In a specific embodiment, an HSPpreparation is administered in conjunction with a non-vaccine treatmentmodality to a subject to reduce the risk of acquiring a type of canceror an infectious disease. In other specific embodiments, the methodsencompass administration of an HSP preparation with administration of anon-vaccine treatment modality as a preventative measure to a subjecthaving a genetic or non-genetic predisposition to a cancer or infectiousdisease or to a subject facing exposure to an agent of an infectiousdisease. In further embodiments, the invention also provides that eachof the foregoing embodiments also can be applicable wherein an α2Mpreparation is administered in conjunction with a non-vaccine treatmentmodality.

The methods and compositions of the invention are useful not only inuntreated patients, but are also useful in the treatment of patientspartially or completely un-responsive to the therapeutic modality in theabsence of the HSP/α2M preparation or to the HSP/α2M preparation in theabsence of the therapeutic modality. In various embodiments, theinvention provides methods and compositions useful in the treatment orprevention of diseases and disorders in patients that have been shown tobe or may be refractory or non-responsive to therapies comprising theadministration of either or both the HSP/α2M preparation or thetherapeutic modality. The invention also includes methods andcompositions comprising administration of the HSP/α2M preparation andthe therapeutic modality to patients that have previously receivedand/or are concurrently receiving other forms of medical therapy.

The HSP preparation used in the methods and compositions of theinvention is preferably purified, and can include free HSP not bound toany molecule, and molecular complexes of HSP with another molecule, suchas a peptide. An HSP-peptide complex comprises an HSP covalently ornoncovalently attached to a peptide. The methods of the invention may ormay not require covalent or noncovalent attachment of an HSP to anyspecific antigens or antigenic peptides prior to administration to asubject. Although, the peptide(s) may be unrelated to the infectiousdisease or disorder or particular cancer being treated, in preferredembodiments, the HSP preparation comprises complexes which display theantigenicity of an antigen of the agent of infectious disease or of atumor specific antigen or tumor associated antigen of the type of cancerbeing treated, respectively. More preferably, for the treatment ofinfectious disease, the HSP preparation comprises noncovalentHSP-peptide complexes isolated from a cell infected with an infectiousagent (or non-infectious variant thereof displaying the antigenicitythereof) that causes the infectious disease. More preferably, fortreatment of a type of cancer, the HSP preparation comprises noncovalentHSP-peptide complexes isolated from cancerous tissue of said type ofcancer or a metastasis thereof, which can be from the patient(autologous) or not (allogeneic). Accordingly, for the purposes of thisinvention, an HSP preparation is a composition comprising HSPs whetherunbound or bound to other molecules (e.g., peptides). The HSP ispreferably purified. An HSP preparation may include crude cell lysatecomprising HSP, the amount of lysate corresponding to between 100 to 10⁸cell equivalents. HSPs can be conveniently purified from most cellularsources as a population of complexes of different peptidesnon-covalently bound to HSPs. The HSPs can be separated from thenon-covalently bound peptides by exposure to low pH and/or adenosinetriphosphate, or other methods known in the art.

The α2M preparation used in the methods and compositions of theinvention is preferably purified, and can include free α2M not bound toany molecule, and molecular complexes of α2M with another molecule, suchas a peptide. An α2M-peptide complex comprises an α2M covalently ornoncovalently attached to a peptide. The methods of the invention may ormay not require covalent or noncovalent attachment of an α2M to anyspecific antigens or antigenic peptides prior to administration to asubject. Although, the peptide(s) may be unrelated to the infectiousdisease or disorder or particular cancer being treated, in preferredembodiments, the α2M preparation comprises complexes which display theantigenicity of an antigen of the agent of infectious disease or of atumor specific antigen or tumor associated antigen of the type of cancerbeing treated, respectively. More preferably, for the treatment ofinfectious disease, the α2M preparation comprises noncovalentα2M-peptide complexes isolated from a cell infected with an infectiousagent (or non-infectious variant thereof displaying the antigenicitythereof) that causes the infectious disease. More preferably, fortreatment of a type of cancer, the α2M preparation comprises noncovalentα2M-peptide complexes isolated from cancerous tissue of said type ofcancer or a metastasis thereof, which can be from the patient(autologous) or not (allogeneic). Accordingly, for the purposes of thisinvention, an α2M preparation is a composition comprising α2M whetherunbound or hound to other molecules (e.g., peptides). The α2M ispreferably purified. An α2M preparation may include crude cell lysatecomprising α2M, the amount of lysate corresponding to between 100 to 10⁸cell equivalents. α2M s can be conveniently purified from most cellularsources as a population of complexes of different peptidesnon-covalently bound to α2Ms. The α2M can be separated from thenon-covalently bound peptides by exposure to low pH and/or adenosinetriphosphate, or other methods known in the art.

In various embodiments, the source of the HSP and the α2M is preferablyan eukaryote, more preferably a mammal, and most preferably a human.Accordingly, the HSP preparation used by the methods of the inventionincludes eukaryotic HSPs, mammalian HSPs and human HSPs. The α2Mpreparation includes eukaryotic α2M, mammalian α2M and human α2M. Theeukaryotic source from which the HSP preparation or α2M preparation isderived and the subject receiving the HSP preparation or the α2Mpreparation, respectively, are preferably the same species.

In one embodiment, the specific immunogenicity of the HSP preparationderives from the peptide complexed to a heat shock protein. Accordingly,in various embodiments, the HSP preparation comprises heat shock proteinpeptide complexes wherein the heat shock proteins are complexed topeptides derived from a specific antigen source. In a preferredembodiment, the HSP protein preparation comprises heat shockprotein-peptide complexes that are autologous. In another preferredembodiment, the HSP preparation comprises heat shock proteins complexedto antigenic peptides of the cancer cells from which they are derived.In specific embodiments, the antigen is a tumor specific antigen (i.e.,only expressed in the tumor cells). In other specific embodiments, theantigen is a tumor associated antigen (i.e., relatively overexpressed inthe tumor cells). In yet another preferred embodiment, the HSPpreparation comprises heat shock proteins complexed to antigenicpeptides of the infected cells from which they are derived.

In another embodiment, the specific immunogenicity of the α2Mpreparation derives from the peptide complexed to an α2M. Accordingly,in various embodiments, the α2M preparation comprises α2M peptidecomplexes wherein the α2M are complexed to peptides derived from aspecific antigen source. In a preferred embodiment, the α2M proteinpreparation comprises α2M-peptide complexes that are autologous. Inanother preferred embodiment, the α2M preparation comprises α2Mcomplexed to antigenic peptides of the cancer cells from which they arederived. In other specific embodiments, the antigen is a tumorassociated antigen (i.e., relatively overexpressed in the tumor cells).In yet another preferred embodiment, the α2M preparation comprises α2Mcomplexed to antigenic peptides of the infected cells from which theyare derived.

Also encompassed by the invention are methods of treatment and delivery,pharmaceutical compositions and formulas comprising administering atleast one non-vaccine therapeutic modality and an HSP preparation or anα2M preparation and kits comprising such pharmaceutical compositions.

4. DESCRIPTION OF THE FIGURE

FIG. 1. Synopsis of clinical protocol described in section 7, infra. Thesynopsis includes all physical examinations, blood work, x-rays and bonemarrow tests that were done before, during and after HSP-peptide complexvaccination.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the recognition that an HSPpreparation can enhance or improve the therapeutic benefit ofnon-vaccine treatment modalities or therapeutic modalities for treatmentof cancer or infectious diseases. Thus, the present inventionencompasses methods and compositions that comprise administering an HSPpreparation in combination with a non-vaccine treatment modality. Alsoencompassed are methods and compositions that comprise administering anα2M preparation in combination with a non-vaccine treatment modality. Inparticular, the invention encompasses methods of treatment andcompositions that provide a better therapeutic profile than that of anHSP preparation or α2M preparation administered alone or a non-vaccinetreatment modality administered alone. The source of the HSP or α2M ispreferably an eukaryote, and most preferably a mammal. The subjectreceiving the treatment is preferably a mammal including, but notlimited to, domestic animals, such as cats and dogs; wild animals,including foxes and raccoons; livestock and fowl, including horses,cattle, sheep, turkeys and chickens, as well as any rodents. Mostpreferably, the subject is human.

The invention provides methods for improving the therapeutic outcome ofa non-vaccine treatment modality comprising administering either an HSPpreparation or an α2M preparation, preferably a purified HSP preparationor a purified α2M preparation, in conjunction with the administration ofthe treatment modality. Either the HSP preparation or the α2Mpreparation can be administered over a period of time which may precede,overlap, and/or follow a treatment regimen with a non-vaccine treatmentmodality. The HSP preparation or the α2M preparation can be administeredconcurrently, before, or after the administration of the treatmentmodality. Examples of treatment modalities include but are not limitedto antibiotics, antivirals, antifungal compounds, anti-cancer treatmentssuch as chemotherapeutic agents, and radiation, as well as biologicaltherapeutic agents and immunotherapeutic agents. In preferredembodiments, the treatment modality is useful in the treatment orprevention of cancer. In another preferred embodiment, the treatmentmodality is Gleevec™.

In one embodiment, the invention encompasses methods of treatment thatprovide better therapeutic profiles than the administration of thetreatment modality or the HSP preparation alone. In another embodiment,the invention encompasses methods of treatment that provide bettertherapeutic profiles than the administration of the treatment modalityor the α2M preparation alone. Encompassed by the invention are methodswherein the administration of a treatment modality with an HSPpreparation or an α2M preparation has additive potency or additivetherapeutic effect. The invention also encompasses synergistic outcomeswhere the therapeutic efficacy is greater than additive. Preferably,such administration of a treatment modality with an HSP preparation orwith an α2M preparation also reduces or avoids unwanted or adverseeffects. Given the invention, in certain embodiments, doses ofnon-vaccine treatment modality can be reduced or administered lessfrequently, preferably increasing patient compliance, improving therapyand/or reducing unwanted or adverse effects. In a specific embodiment,lower or less frequent doses of chemotherapy or radiation therapy areadministered to reduce or avoid unwanted effects. Alternatively, dosesof HSP preparation and doses of α2M preparation can be reduced oradministered less frequently if administered with a treatment modality.

In one embodiment, the present invention provides a method for improvingthe outcome of a treatment in a subject receiving a therapeutic modalitywhich is not a vaccine. The method comprises administering either a heatshock protein preparation, preferably a purified HSP preparation, or anα2M preparation, preferably a purified α2M preparation, to the subjectbefore, concurrently with, or after the administration of thetherapeutic modality. In a specific embodiment, the HSP preparation orthe α2M preparation can augment the therapeutic benefit of a treatmentmodality and improve the outcome of the treatment. Without being boundby any theory or mechanism, the administration of a mammalian HSPpreparation or α2M preparation to a subject can enhance theresponsiveness of non-specific immune mechanisms of the subject, forexample, by increasing the number of natural killer (NK) cells and/oraccelerating the maturation of dendritic cells and/or can also enhancethe responsiveness of specific immune mechanisms, such as by increasingthe number of CD4+ and CD8+ T cells. In a specific embodiment, the HSPpreparation is administered before the administration of the therapeuticmodality. In another specific embodiment, the therapeutic modality isadministered before the administration of the HSP preparation. Inspecific embodiment, the α2M preparation is administered before theadministration of the therapeutic modality. In another specificembodiment, the therapeutic modality is administered before theadministration of the α2M preparation.

In another embodiment, the present invention provides a method forimproving the outcome of a treatment in a subject receiving an HSPpreparation, preferably a purified HSP preparation, by administering anon-vaccine therapeutic modality to the subject before, concurrentlywith, or after the administration of the HSP preparation. In a specificembodiment, the non-vaccine therapeutic modality can augment thetherapeutic benefit of an HSP preparation and improve the outcome of thetreatment.

In another embodiment, the present invention provides a method forimproving the outcome of a treatment in a subject receiving an α2Mpreparation, preferably a purified α2M preparation, by administering anon-vaccine therapeutic modality to the subject before, concurrentlywith, or after the administration of the α2M preparation. In a specificembodiment, the non-vaccine therapeutic modality can augment thetherapeutic benefit of an α2M preparation and improve the outcome of thetreatment.

In certain embodiments, the administration of the HSP/α2M preparation inthe absence of administration of the therapeutic modality or theadministration of the therapeutic modality in the absence ofadministration of the HSP/α2M preparation is not therapeuticallyeffective. In a specific embodiment, the amount of HSP/α2M preparationor therapeutic modality is administered in an amount insufficient to betherapeutically effective alone. In alternate embodiments, both or atleast one of the HSP/α2M preparation or therapeutic modality istherapeutically effective when administered alone.

In various embodiments, the methods comprise the administration of anHSP preparation, preferably a purified HSP preparation, to a subjectreceiving a treatment modality for the treatment of cancer or infectiousdiseases. Preferably the HSP preparation comprises HSP-peptide complexesdisplaying the antigenicity of a tumor specific antigen or tumorassociated antigen of the type of cancer or an antigen of an infectiousagent, i.e., heat shock proteins complexed to antigenic peptides of thecancer cells or infected cells from which the complexes are obtained.Accordingly, in one embodiment, the specific immunogenicity of the HSPpreparation derives from the peptide complexed to the HSP. In preferredembodiments, the HSP-peptide complexes are isolated from an antigensource such as cancer tissues or infected tissues. In the practice ofthe invention, such HSP-peptide complexes are preferably, autologous tothe individual subject, i.e., obtained from the tissues of the subjectreceiving the administration of HSP preparation and treatment modality,but need not be (i.e., allogeneic to the individual subject).

In various other embodiments, the methods comprise the administration ofan α2M preparation, preferably a purified α2M preparation, to a subjectreceiving a treatment modality for the treatment of cancer or infectiousdiseases. Preferably the α2M preparation comprises α2M-peptide complexesdisplaying the antigenicity of a tumor specific antigen or tumorassociated antigen of the type of cancer or an antigen of an infectiousagent, i.e., α2M complexed to antigenic peptides of the cancer cells orinfected cells from which the complexes are obtained. Accordingly, inone embodiment, the specific immunogenicity of the α2M preparationderives from the peptide complexed to the α2M. In preferred embodiments,the α2M-peptide complexes are isolated from an antigen source such ascancer tissues or infected tissues. In the practice of the invention,such α2M-peptide complexes are preferably, autologous to the individualsubject, i.e., obtained from the tissues of the subject receiving theadministration of α2M preparation and treatment modality, but need notbe (i.e., allogeneic to the individual subject).

In one embodiment, the methods comprise the administration of an HSPpreparation or an α2M preparation, preferably a purified HSP preparationor a purified α2M preparation, to a subject receiving a treatmentmodality for treatment of an infectious disease. Such treatmentmodalities are known in the art and include but are not limited toantibiotics, antivirals, antifungals as well as biological andimmunotherapeutic agents. Preferably the HSP preparation comprisesHSP-peptide complexes which display the antigenicity of an agent of theinfectious disease. Preferably the α2M preparation comprises α2M-peptidecomplexes which display the antigenicity of an agent of the infectiousdisease. In a specific embodiment, the outcome of a treatment of a typeof infectious disease in a subject receiving a non-vaccine therapeuticmodality is improved by administering HSP-peptide complexes comprisingan HSP complexed to a peptide that displays the antigenicity of anantigen of an agent of said type of infectious disease. Preferably, theHSP-peptide complexes are not present in admixture with HSP or α2M thatis not complexed to a peptide that displays the antigenicity of anantigen of an agent of the same infectious disease. (See InternationalApplication No. PCT/US01/28840, filed Sep. 15, 2001). In one embodiment,the HSP preparation is administered prior to administration of thetherapeutic modality. In another embodiment, the therapeutic modality isadministered prior to administration of the HSP preparation. In anotherspecific embodiment, the outcome of a treatment of a type of infectiousdisease in a subject receiving a non-vaccine therapeutic modality isimproved by administering α2M-peptide complexes comprising an α2Mcomplexed to a peptide that displays the antigenicity of an antigen ofan agent of said type of infectious disease. Preferably, the α2M-peptidecomplexes are not present in admixture with HSP or α2M that is notcomplexed to a peptide that displays the antigenicity of an antigen ofan agent of the same infectious disease. Preferably, the α2M preparationis administered prior to administration of the therapeutic modality.

In another embodiment, the methods comprise the administration of eitheran HSP preparation or an α2M preparation, preferably a purified HSPpreparation or a purified α2M preparation, to a subject receiving atreatment modality for treatment of cancer. Such treatment modalitiesinclude but are not limited to anti-cancer therapies such aschemotherapies and radiation therapies as well as hormonal therapies,biological therapies and immunotherapies. In the methods of theinvention the anti-cancer agents that can be used include but are notlimited to cytotoxic agents, antimitotic agents, tubulin stabilizingagents, microtubule formation inhibiting agents, topoisomerase activeagents, alkylating agents, DNA interactive agents, antimetabolites,RNA/DNA antimetabolites, and DNA antimetabolites. Preferably, theanti-cancer agent is a chemotherapeutic agent. Preferably the HSPpreparation or α2M preparation is administered to a subject receiving achemotherapy or radiation therapy for treatment of cancer. Preferablythe HSP preparation comprises HSP-peptide complexes which display theantigenicity of the type of cancer being treated. Preferably where thepreparation is an α2M preparation, the α2M preparation comprisesα2M-peptide complexes which display the antigenicity of the type ofcancer being treated. Accordingly, in preferred embodiments, theinvention provides methods for improving the outcome of cancer treatmentin a subject receiving a therapeutic modality which is not a vaccineusing HSP-peptide complexes comprising an HSP complexed to a peptidethat displays the antigenicity of a tumor specific antigen or tumorassociated antigen of a type of cancer or using α2M-peptide complexescomprising an α2M complexed to a peptide that displays the antigenicityof a tumor specific antigen or tumor associated antigen of a type ofcancer. In certain preferred embodiments, such HSP-peptide complexes andα2M-peptide complexes are not diluted with either HSP or α2M that is notcomplexed to a peptide that displays the antigenicity of an antigen ofthe same type of cancer. Preferably, the HSP preparation or α2Mpreparation is administered prior to administration of the therapeuticmodality.

In a specific embodiment, an HSP preparation is administered to asubject receiving a chemotherapeutic agent for treatment of cancer. Inanother preferred embodiment, an α2M preparation is administered to asubject receiving a chemotherapeutic agent for treatment of cancer. Suchchemotherapeutic agents are known in the art and include but are notlimited to: methotrexate, taxol, mercaptopurine, thioguanine,hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas,cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine,etoposides, campathecins, bleomycin, doxorubicin, idarubicin,daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase,vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel,doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel andpaclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide,nitrosoureas such as carmustine and lomustine, vinca alkaloids, platinumcompounds, mitomycin, gemcitabine, hexamethylmelamine, topotecan,tyrosine kinase inhibitors, tyrphostins, Gleevec™ (imatinib mesylate),herbimycin A, genistein, erbstatin, and lavendustin A. In a preferredembodiment, the chemotherapeutic agent is Gleevec™ (imatinib mesylate).

In other embodiments, suitable chemotherapeutics include, but are notlimited to, methotrexate, taxol, L-asparaginase, mercaptopurine,thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine,procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide,5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin,idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone,asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, anddocetaxel. In a preferred embodiment, the anti-cancer agent can be, butis not limited to, a drug listed in Table 1.

TABLE 1 Alkylating agents Nitrogen mustards: Cyclophosphamide IfosfamideTrofosfamide Chlorambucil Nitrosoureas: Carmustine (BCNU) Lomustine(CCNU) Alkylsulphonates: Busulfan Treosulfan Triazenes: DacarbazinePlatinum containing Cisplatin compounds: Carboplatin AroplatinOxaliplatin Plant Alkaloids Vinca alkaloids: Vincristine VinblastineVindesine Vinorelbine Taxoids: Paclitaxel Docetaxel DNA TopoisomeraseInhibitors Epipodophyllins: Etoposide Teniposide Topotecan9-aminocamptothecin Camptothecin Crisnatol mitomycins: Mitomycin CAnti-metabolites Anti-folates: DHFR inhibitors: MethotrexateTrimetrexate IMP dehydrogenase Mycophenolic acid Inhibitors: TiazofurinRibavirin EICAR Ribonuclotide reductase Hydroxyurea Inhibitors:Deferoxamine Pyrimidine analogs: Uracil analogs: 5-FluorouracilFloxuridine Doxifluridine Ratitrexed Cytosine analogs: Cytarabine (araC) Cytosine arabinoside Fludarabine Purine analogs: MercaptopurineThioguanine DNA Antimetabolites: 3-HP 2′-deoxy-5-fluorouridine 5-HPalpha-TGDR aphidicolin glycinate ara-C 5-aza-2′-deoxycytidine beta-TGDRcyclocytidine guanazole inosine glycodialdehyde macebecin IIpyrazoloimidazole Hormonal therapies: Receptor antagonists:Anti-estrogen: Tamoxifen Raloxifene Megestrol LHRH agonists: GoserelinLeuprolide acetate Anti-androgens: Flutamide BicalutamideRetinoids/Deltoids Cis-retinoic acid Vitamin A derivative: All-transretinoic acid (ATRA-IV) Vitamin D3 analogs: EB 1089 CB 1093 KH 1060Photodynamic therapies: Vertoporfin (BPD-MA) PhthalocyaninePhotosensitizer Pc4 Demethoxy-hypocrellin A (2BA-2-DMHA) Cytokines:Interferon-α Interferon-γ Tumor necrosis factor Angiogenesis Inhibitors:Angiostatin (plasminogen fragment) antiangiogenic antithrombin IIIAngiozyme ABT-627 Bay 12-9566 Benefin Bevacizumab BMS-275291cartilage-derived inhibitor (CDI) CAI CD59 complement fragment CEP-7055Col 3 Combretastatin A-4 Endostatin (collagen XVIII fragment)Fibronectin fragment Gro-beta Halofuginone Heparinases Heparinhexasaccharide fragment HMV833 Human chorionic gonadotropin (hCG) IM-862Interferon alpha/beta/gamma Interferon inducible protein (IP-10)Interleukin-12 Kringle 5 (plasminogen fragment) MarimastatMetalloproteinase inhibitors (TIMPs) 2-Methoxyestradiol MMI 270 (CGS27023A) MoAb IMC-1C11 Neovastat NM-3 Panzem PI-88 Placental ribonucleaseinhibitor Plasminogen activator inhibitor Platelet factor-4 (PF4)Prinomastat Prolactin 16 kD fragment Proliferin-related protein (PRP)PTK 787/ZK 222594 Retinoids Solimastat Squalamine SS 3304 SU 5416 SU6668SU11248 Tetrahydrocortisol-S tetrathiomolybdate thalidomideThrombospondin-1 (TSP-1) TNP-470 Transforming growth factor-beta (TGF-b)Vasculostatin Vasostatin (calreticulin fragment) ZD6126 ZD 6474 farnesyltransferase inhibitors (FTI) bisphosphonates Antimitotic agents:allocolchicine Halichondrin B colchicine colchicine derivative dolstatin10 maytansine rhizoxin thiocolchicine trityl cysteine Others:Isoprenylation inhibitors: Dopaminergic neurotoxins:1-methyl-4-phenylpyridinium ion Cell cycle inhibitors: StaurosporineActinomycins: Actinomycin D Dactinomycin Bleomycins: Bleomycin A2Bleomycin B2 Peplomycin Anthracyclines: Daunorubicin Doxorubicin(adriamycin) Idarubicin Epirubicin Pirarubicin Zorubicin MitoxantroneMDR inhibitors: Verapamil Ca²⁺ATPase inhibitors: Thapsigargin

Additional anti-cancer agents that may be used in the methods of thepresent invention include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin U, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other anti-cancer drugs that can be used include, but are not limitedto: 20-epi-1,25 dihydroxyvitamin 133; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustardanti-cancer agent; mycaperoxide B; mycobacterial cell wall extract;myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin;nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim;nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant;nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides;onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxelanalogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor, stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred chemotherapeutics of the invention includeGleevec™ (imatinib mesylate) and other tyrosine kinase inhibitors.

In preferred embodiments, each of the methods above compriseadministering either an HSP preparation or an α2M preparation,preferably a purified HSP preparation or a purified α2M preparation, toa subject receiving a drug of the 2-phenylaminopyrimidine class fortreatment of cancer. More preferably, the subject is receiving Gleevec™(i.e., imatinib mesylate) for treatment of cancer.

In another preferred embodiment, an HSP preparation or an α2Mpreparation is administered to a subject receiving radiation therapy fortreatment of cancer. For radiation treatment, the radiation can be gammarays or X-rays. The methods encompass treatment of cancer comprisingradiation therapy, such as external-beam radiation therapy, interstitialimplantation of radioisotopes (I-125, palladium, iridium), radioisotopessuch as strontium-89, thoracic radiation therapy, intraperitoneal P-32radiation therapy, and/or total abdominal and pelvic radiation therapy.For a general overview of radiation therapy, see Hellman, Chapter 16:Principles of Cancer Management: Radiation Therapy, 6th edition, 2001,DeVita et al., eds., J.B. Lippencott Company, Philadelphia. In preferredembodiments, the radiation treatment is administered as external beamradiation or teletherapy wherein the radiation is directed from a remotesource. In various preferred embodiments, the radiation treatment isadministered as internal therapy or brachytherapy wherein a radioactivesource is placed inside the body close to cancer cells or a tumor mass.

In another embodiment, the each of the above methods comprise theadministration of HSP preparation, preferably a purified HSPpreparation, to a subject receiving a combination of treatmentmodalities for the treatment of cancer. In another embodiment, the eachof the above methods comprise the administration of an α2M preparation,preferably a purified α2M preparation, to a subject receiving acombination of treatment modalities for the treatment of cancer.Preferably the HSP preparation and α2M preparation each comprisesHSP-peptide complexes and α2M-peptide complexes, respectively, whichdisplay the antigenicity of the type of cancer being treated. In onesuch embodiment, HSP preparation is administered to a subject receivinga chemotherapy in combination with a biological therapy, preferably acytokine. In another such embodiment, an α2M preparation is administeredto a subject receiving a chemotherapy in combination with a biologicaltherapy, preferably a cytokine. In various embodiments, the cytokine isselected from the group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFNα, IFNβ, IFNγ,TNFα, TNFβ, G-CSF, GM-CSF, TGF-β, IL-15, IL-18, GM-CSF, INF-γ, SLC,endothelial monocyte activating protein-2 (EMAP2), MIP-3α, MIP-3β, or anMHC gene, such as HLA-B7. Additionally, other exemplary cytokinesinclude other members of the TNF family, including but not limited toTNF-α-related apoptosis-inducing ligand (TRAIL), TNF-α-relatedactivation-induced cytokine (TRANCE), TNF-α-related weak inducer ofapoptosis (TWEAK), CD40 ligand (CD40L), LT-α, LT-β, OX4OL, CD4OL, FasL,CD27L, CD30L, 4-1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17, and AITR-L,or a functional portion thereof. See, e.g., Kwon et al., 1999, Curr.Opin. Immunol. 11:340-345 for a general review of the TNF family.Preferably, the HSP preparation is administered prior to the treatmentmodalities.

In a specific embodiment, a purified HSP preparation is administered toa subject receiving cyclophosphamide in combination with IL-12 fortreatment of cancer. In another specific embodiment, a purified α2Mpreparation is administered to a subject receiving cyclophosphamide incombination with IL-12 for treatment of cancer.

In another specific embodiment, the chemotherapeutic is a tyrosinekinase inhibitor, the HSP preparation is obtained from the cancersubject being treated, and the chemotherapy is administered prior toadministration of the HSP preparation. In another specific embodiment,the anti-cancer agent is the chemotherapeutic Gleevec™ (imatinibmesylate), the HSP preparation comprises hsp70 obtained from the cancersubject being treated, and the chemotherapeutic is administered prior toadministration of the HSP preparation. In another specific embodiment,the HSP preparation comprises hsp70-peptide complexes obtained from thecancer subject being treated. Another specific embodiment encompasses amethod for treating CML in a subject receiving about 400 mg to 800 mg ofimatinib mesylate daily comprising administering a heat shock proteinpreparation to said subject, wherein said heat shock protein preparationcomprises hsp70 peptide complexes. In preferred embodiments, the heatshock protein preparation is administered once a week and the heat shockprotein preparation comprises hsp70-peptide complexes obtained from saidsubject.

In certain specific embodiments, an HSP preparation is administered to asubject already receiving Gleevec™ (e.g., 400-800 mg daily in capsuleform, 400-600 mg doses administered once daily, or 800 mg doseadministered daily in two doses of 400 mg each). In such embodiments, anHSP/α2M preparation is initially administered to a subject who hasalready been receiving Gleevec™ in the absence of HSP/α2M preparation 2days, 2 days to 1 week, 1 week to 1 month, 1 month to 6 months, 6 monthsto 1 year prior to administration of HSP/α2M preparation in addition toGleevec™. In a specific embodiment, an HSP/α2M preparation isadministered to a subject wherein the subject showed resistance totreatment with Gleevec™ alone.

In other embodiments, an HSP/α2M preparation is initially administeredto a subject concurrently with the initial administration of Gleevec™.

In yet other specific embodiments, Gleevec™ (e.g., 400-800 mg daily incapsule form) is administered to a subject already receiving treatmentcomprising administration of an HSP/α2M preparation. In suchembodiments, Gleevec™ is initially administered to a subject who hasalready been receiving an HSP/α2M preparation in the absence of Gleevec™2 days, 2 days to 1 week, 1 week to 1 month, 1 month to 6 months, 6months to 1 year prior to administration of Gleevec™ in addition toadministration of an HSP/α2M preparation.

In a specific embodiment, Gleevec™ is administered orally. In anotherspecific embodiment, the HSP preparation is administered intradermally.

In each of the methods contemplated above, the patient, by way ofexample, receives 50 mg to 100 mg, 100 mg to 200 mg, 200 mg to 300 mg,300 mg to 400 mg, 400 mg to 500 mg, 500 mg to 600 mg, 600 mg to 700 mg,700 mg to 800 mg, 800 mg to 900 mg, or 900 mg to 1000 mg of Gleevec™daily. In certain embodiments, the total daily dose is administered to asubject as two daily doses of 25 mg to 50 mg, 50 mg to 100 mg, 100 mg to200 mg, 200 mg to 300 mg, 300 mg to 400 mg, or 400 mg to 500 mg.

Other treatment modalities contemplated include but are not limited toantiviral agents known in the art. Such antiviral agents include but arenot limited to: ribavirin, rifampicin, AZT, ddI, ddC, acyclovir andganciclovir.

Also encompassed by the invention are therapeutic modalities that areantibiotic agents known in the art including but not limited to:aminoglycoside antibiotics apramycin, arbekacin, bambermycins,butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin,paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicolantibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, andthiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin),carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem andimipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, andcefpirome), cephamycins (e.g., cefbuperazone, cefmetazole, andcefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam),oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g.,amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,benzylpenicillinic acid, benzylpenicillin sodium, epicillin,fenbenicillin, floxacillin, penamecillin, penethamate hydriodide,penicillin o-benethamine, penicillin 0, penicillin V, penicillin Vbenzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), macrolides (e.g., azithromycin, carbomycin, clarithomycin,dirithromycin, erythromycin, and erythromycin acistrate), amphomycin,bacitracin, capreomycin, colistin, enduracidin, enviomycin,tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, anddemeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans(e.g., furaltadone, and furazolium chloride), quinolones and analogsthereof (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine, andgrepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine,benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone,glucosulfone sodium, and solasulfone), cycloserine, mupirocin andtuberin.

Also encompassed by the invention are therapeutic modalities that areantifungal agents and known in the art and include but are not limitedto: polyenes (e.g., amphotericin b, candicidin, mepartricin, natamycin,and nystatin), allylamines (e.g., butenafine, and naftifine), imidazoles(e.g., bifonazole, butoconazole, chlordantoin, flutrimazole,isoconazole, ketoconazole, and lanoconazole), thiocarbamates (e.g.,tolciclate, tolindate, and tolnaftate), triazoles fluconazole,itraconazole, saperconazole, and terconazole), bromosalicylchloranilide,buclosamide, calcium propionate, chlorphenesin, ciclopirox, azaserine,griseofulvin, oligomycins, neomycin undecylenate, pyrrolnitrin,siccanin, tubercidin, and viridin.

In another embodiment, the above methods are useful for the preventionof cancer or infectious disease. In a specific embodiment, an HSPpreparation is administered in conjunction with a non-vaccine treatmentmodality to a subject to reduce the risk of acquiring a type of canceror an infectious disease. In other specific embodiments, the methodsencompass administration of an HSP preparation with administration of anon-vaccine treatment modality as a preventative measure to a subjecthaving a genetic or non-genetic predisposition to a cancer or infectiousdisease or to a subject facing exposure to an agent of an infectiousdisease. In further embodiments, the invention also provides that eachof the foregoing embodiments also can be applicable wherein an α2Mpreparation is administered in conjunction with a non-vaccine treatmentmodality.

The methods and compositions of the invention are useful not only inuntreated patients, but are also useful in the treatment of patientspartially or completely un-responsive to the therapeutic modality in theabsence of the HSP/α2M preparation or to the HSP/α2M preparation in theabsence of the therapeutic modality. In various embodiments, theinvention provides methods and compositions useful in the treatment orprevention of diseases and disorders in patients that have been shown tobe or may be refractory or non-responsive to therapies comprising theadministration of either or both the HSP/α2M preparation or thetherapeutic modality. The invention also includes methods andcompositions comprising administration of the HSP/α2M preparation andthe therapeutic modality to patients that have previously receivedand/or are concurrently receiving other forms of medical therapy.

The HSP preparation used in the methods and compositions of theinvention is preferably purified, and can include free HSP not bound toany molecule, and molecular complexes of HSP with another molecule, suchas a peptide. An HSP-peptide complex comprises an HSP covalently ornoncovalently attached to a peptide. The methods of the invention may ormay not require covalent or noncovalent attachment of an HSP to anyspecific antigens or antigenic peptides prior to administration to asubject. Although, the peptide(s) may be unrelated to the infectiousdisease or disorder or particular cancer being treated, in preferredembodiments, the HSP preparation comprises complexes which display theantigenicity of an antigen of the agent of infectious disease or of atumor specific antigen or tumor associated antigen of the type of cancerbeing treated, respectively. More preferably, for the treatment ofinfectious disease, the HSP preparation comprises noncovalentHSP-peptide complexes isolated from a cell infected with an infectiousagent (or non-infectious variant thereof displaying the antigenicitythereof) that causes the infectious disease. More preferably, fortreatment of a type of cancer, the HSP preparation comprises noncovalentHSP-peptide complexes isolated from cancerous tissue of said type ofcancer or a metastasis thereof, which can be from the patient(autologous) or not (allogeneic). Accordingly, for the purposes of thisinvention, an HSP preparation is a composition comprising HSPs whetherunbound or bound to other molecules (e.g., peptides). The HSP ispreferably purified. An HSP preparation may include crude cell lysatecomprising HSP, the amount of lysate corresponding to between 100 to 10⁸cell equivalents. HSPs can be conveniently purified from most cellularsources as a population of complexes of different peptidesnon-covalently bound to HSPs. The HSPs can be separated from thenon-covalently bound peptides by exposure to low pH and/or adenosinetriphosphate, or other methods known in the art.

The α2M preparation used in the methods and compositions of theinvention is preferably purified, and can include free α2M not bound toany molecule, and molecular complexes of α2M with another molecule, suchas a peptide. An α2M-peptide complex comprises an α2M covalently ornoncovalently attached to a peptide. The methods of the invention may ormay not require covalent or noncovalent attachment of an α2M to anyspecific antigens or antigenic peptides prior to administration to asubject. Although, the peptide(s) may be unrelated to the infectiousdisease or disorder or particular cancer being treated, in preferredembodiments, the α2M preparation comprises complexes which display theantigenicity of an antigen of the agent of infectious disease or of atumor specific antigen or tumor associated antigen of the type of cancerbeing treated, respectively. More preferably, for the treatment ofinfectious disease, the α2M preparation comprises noncovalentα2M-peptide complexes isolated from a cell infected with an infectiousagent (or non-infectious variant thereof displaying the antigenicitythereof) that causes the infectious disease. More preferably, fortreatment of a type of cancer, the α2M preparation comprises noncovalentα2M-peptide complexes isolated from cancerous tissue of said type ofcancer or a metastasis thereof, which can be from the patient(autologous) or not (allogeneic). Accordingly, for the purposes of thisinvention, an α2M preparation is a composition comprising α2M whetherunbound or bound to other molecules (e.g., peptides). The α2M ispreferably purified. An α2M preparation may include crude cell lysatecomprising α2M, the amount of lysate corresponding to between 100 to 10⁸cell equivalents. α2M s can be conveniently purified from most cellularsources as a population of complexes of different peptidesnon-covalently bound to α2Ms. The α2M can be separated from thenon-covalently bound peptides by exposure to low pH and/or adenosinetriphosphate, or other methods known in the art.

In various embodiments, the source of the HSP and the α2M is preferablyan eukaryote, more preferably a mammal, and most preferably a human.Accordingly, the HSP preparation used by the methods of the inventionincludes eukaryotic HSPs, mammalian HSPs and human HSPs. The α2Mpreparation includes eukaryotic α2M, mammalian α2M and human α2M. Theeukaryotic source from which the HSP preparation or α2M preparation isderived and the subject receiving the HSP preparation or the α2Mpreparation, respectively, are preferably the same species.

In one embodiment, the specific immunogenicity of the HSP preparationderives from the peptide complexed to a heat shock protein. Accordingly,in various embodiments, the HSP preparation comprises heat shock proteinpeptide complexes wherein the heat shock proteins are complexed topeptides derived from a specific antigen source. In a preferredembodiment, the HSP protein preparation comprises heat shockprotein-peptide complexes that are autologous. In another preferredembodiment, the HSP preparation comprises heat shock proteins complexedto antigenic peptides of the cancer cells from which they are derived.In specific embodiments, the antigen is a tumor specific antigen (i.e.,only expressed in the tumor cells). In other specific embodiments, theantigen is a tumor associated antigen (i.e., relatively overexpressed inthe tumor cells). In yet another preferred embodiment, the HSPpreparation comprises heat shock proteins complexed to antigenicpeptides of the infected cells from which they are derived.

In another embodiment, the specific immunogenicity of the α2Mpreparation derives from the peptide complexed to an α2M. Accordingly,in various embodiments, the α2M preparation comprises α2M peptidecomplexes wherein the α2M are complexed to peptides derived from aspecific antigen source. In a preferred embodiment, the α2M proteinpreparation comprises α2M-peptide complexes that are autologous. Inanother preferred embodiment, the α2M preparation comprises α2Mcomplexed to antigenic peptides of the cancer cells from which they arederived. In other specific embodiments, the antigen is a tumorassociated antigenic (i.e., relatively overexpressed in the tumorcells). In yet another preferred embodiment, the α2M preparationcomprises α2M complexed to antigenic peptides of the infected cells fromwhich they are derived.

In various specific embodiments, the above methods comprise theadministration of HSP preparation or α2M preparation to a subjecttreated with a treatment modality wherein the treatment modalityadministered alone is not clinically adequate to treat the subject suchthat the subject needs additional effective therapy, e.g., a subject isunresponsive to a treatment modality without administering HSPpreparation or α2M preparation. Included in such embodiments are methodscomprising administering HSP preparation or α2M preparation to a subjectreceiving a treatment modality wherein said subject has responded totherapy yet suffers from side effects, relapse, develops resistance,etc. Such a subject might be non-responsive or refractory to treatmentwith the treatment modality alone. The embodiments provide that themethods of the invention comprising administration of HSP preparation toa subject refractory to a treatment modality alone can improve thetherapeutic effectiveness of the treatment modality when administered ascontemplated by the methods of the invention. The methods of theinvention comprising administration of an α2M preparation to a subjectrefractory to a treatment modality alone can also improve thetherapeutic effectiveness of the treatment modality when administered ascontemplated by the methods of the invention.

In a specific embodiment, an HSP preparation is administered to asubject receiving a treatment modality for the treatment of cancerwherein the subject may be non-responsive or refractory to treatmentwith the treatment modality alone, i.e., at least some significantportion of cancer cells are not killed or their cell division is notarrested. The determination of the effectiveness of a treatment modalitycan be assayed in vivo or in vitro using methods known in the art.Art-accepted meanings of refractory are well known in the context ofcancer. In one embodiment, a cancer is refractory or non-responsivewhere the number of cancer cells has not been significantly reduced, orhas increased. In a preferred embodiment, an HSP preparation thatdisplays the antigenicity of a type of cancer is administered to asubject non-responsive to administration of a treatment modality alone,wherein the administration of HSP preparation improves the effectivenessof the treatment modality. Among these subjects being treated are thosereceiving chemotherapy or radiation therapy.

In a specific embodiment, an α2M preparation is administered to asubject receiving a treatment modality for the treatment of cancerwherein the subject may be non-responsive or refractory to treatmentwith the treatment modality alone, i.e., at least some significantportion of cancer cells are not killed or their cell division is notarrested. The determination of the effectiveness of a treatment modalitycan be assayed in vivo or in vitro using methods known in the art.Art-accepted meanings of refractory are well known in the context ofcancer. In one embodiment, a cancer is refractory or non-responsivewhere the number of cancer cells has not been significantly reduced, orhas increased. In a preferred embodiment, an α2M preparation thatdisplays the antigenicity of a type of cancer is administered to asubject non-responsive to administration of a treatment modality alone,wherein the administration of α2M preparation improves the effectivenessof the treatment modality. Among these subjects being treated are thosereceiving chemotherapy or radiation therapy.

In a specific embodiment, an HSP preparation is administered to asubject receiving a treatment modality for the treatment of cancerwherein the subject may experience unwanted or adverse effects totreatment with the treatment modality alone, e.g., the treatmentmodality may be toxic or harmful at its effective dose, administeredalone. Given the invention, the HSP preparation can improve thetherapeutic benefit of the treatment modality such that the dosage orfrequency of administration of the treatment modality can be loweredwhen administered in conjunction with HSP preparation. In a preferredembodiment, an HSP preparation that displays the antigenicity of a typeof cancer is administered to a subject to reduce or avoid the unwantedor adverse effects of a treatment modality alone, wherein theadministration of HSP preparation allows lower and/or less frequentdoses of the treatment modality. Among these subjects being treated arethose receiving chemotherapy or radiation therapy.

In a specific embodiment, an α2M preparation is administered to asubject receiving a treatment modality for the treatment of cancerwherein the subject may experience unwanted or adverse effects totreatment with the treatment modality alone, e.g., the treatmentmodality may be toxic or harmful at its effective dose, administeredalone. Given the invention, the α2M preparation can improve thetherapeutic benefit of the treatment modality such that the dosage orfrequency of administration of the treatment modality can be loweredwhen administered in conjunction with α2M preparation. In a preferredembodiment, an α2M preparation that displays the antigenicity of a typeof cancer is administered to a subject to reduce or avoid the unwantedor adverse effects of a treatment modality alone, wherein theadministration of α2M preparation allows lower and/or less frequentdoses of the treatment modality. Among these subjects being treated arethose receiving chemotherapy or radiation therapy.

In a specific embodiment, the HSP preparation is administered in asub-optimal amount, e.g., an amount that does not manifest detectabletherapeutic benefits when administered in the absence of the therapeuticmodality, as determined by methods known in the art. In such methods,the administration of such a sub-optimal amount of HSP preparation to asubject receiving a therapeutic modality results in an overallimprovement in effectiveness of treatment. In another specificembodiment, the α2M preparation is administered in a sub-optimal amount.In such methods, the administration of such a sub-optimal amount of α2Mpreparation to a subject receiving a therapeutic modality results in anoverall improvement in effectiveness of treatment.

In a preferred embodiment, an HSP preparation is administered in anamount that does not result in tumor regression or cancer remission oran amount wherein the cancer cells have not been significantly reducedor have increased when said HSP preparation is administered in theabsence of the therapeutic modality. Preferably the HSP preparationcomprises HSP-peptide complexes displaying the antigenicity of thecancer type being treated. In a preferred embodiment, the sub-optimalamount of HSP preparation is administered to a subject receiving atreatment modality whereby the overall effectiveness of treatment isimproved. In another preferred embodiment, an α2M preparation isadministered in an amount that does not result in tumor regression orcancer remission or an amount wherein the cancer cells have not beensignificantly reduced or have increased when said α2M preparation isadministered in the absence of the therapeutic modality. Preferably theα2M preparation comprises α2M-peptide complexes displaying theantigenicity of the cancer type being treated. In a preferredembodiment, the sub-optimal amount of α2M preparation is administered toa subject receiving a treatment modality whereby the overalleffectiveness of treatment is improved. Among these subjects beingtreated with HSP or α2M preparation are those receiving chemotherapy orradiation therapy. A sub-optimal amount can be determined by appropriateanimal studies. Such a sub-optimal amount in humans can be determined byextrapolation from experiments in animals.

The HSP preparation or α2M preparation can be administered prior to,concurrently with, or subsequent to the administration of thenon-vaccine treatment modality. In one embodiment, the HSP preparationand therapeutic modality are administered at exactly the same time. Inanother embodiment, the α2M preparation and therapeutic embodiment areadministered at exactly the same time. In another embodiment the eitherthe HSP, preparation or the α2M preparation and treatment modality areadministered in a sequence and within a time interval such that the HSPpreparation and treatment modality can act together to provide anincreased benefit than if they were administered alone or such that theα2M preparation and treatment modality can act together to provide anincreased benefit than if they were administered alone. In anotherembodiment, the HSP preparation and treatment modality are administeredsufficiently close in time so as to provide the desired therapeuticoutcome. In another embodiment, the α2M preparation and treatmentmodality are administered sufficiently close in time so as to providethe desired therapeutic outcome. The HSP or α2M preparation and thetherapeutic modality can be administered simultaneously or separately,in any appropriate form and by any suitable route. In one embodiment,the HSP preparation and treatment modality are administered by differentroutes of administration. In an alternate embodiment, each isadministered by the same route of administration. The HSP preparationcan be administered at the same or different sites, e.g. arm and leg. Inanother embodiment, the α2M preparation and treatment modality areadministered by different routes of administration. Alternatively, eachcan be administered by the same route. In addition, each could beadministered at the same or different sites.

In various embodiments, such as those described above, the HSPpreparation and treatment modality are administered less than 1 hourapart, at about 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hoursto 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hoursapart, 11 hours to 12 hours apart, no more than 24 hours apart or nomore than 48 hours apart, or no more than 1 week or 2 weeks or 1 monthor 3 months apart. In other embodiments, the HSP preparation andtreatment modality are administered 2 to 4 days apart, 4 to 6 daysapart, 1 week apart, 1 to 2 weeks apart, 2 to 4 weeks apart, one monthapart, 1 to 2 months apart, or 2 or more months apart. In preferredembodiments, the HSP preparation and treatment modality are administeredin a time frame where both are still active. One skilled in the artwould be able to determine such a time frame by determining the halflife of each administered component. In separate or in the foregoingembodiments, the HSP preparation and treatment modality are administeredless than 2 weeks, one month, six months, 1 year or 5 years apart.Preferably, the HSP preparation is administered prior to the treatmentmodality. In further embodiments, the α2M preparation and treatmentmodality are administered at the time intervals and time framesdescribed in each of the above embodiments. Preferably the α2Mpreparation is administered prior to the treatment modality. Preferably,in each of the above embodiments, the treatment modality is acombination of a chemotherapy and cytokine treatment.

In one embodiment, the treatment modality is administered daily and theHSP preparation or α2M preparation is administered once a week for thefirst 4 weeks, and then once every other week thereafter. In oneembodiment, the treatment modality is administered daily and the HSPpreparation or α2M preparation is administered once a week for the first8 weeks, and then once every other week thereafter.

In one embodiment, two or more components are administered within thesame patient visit. In one embodiment, the α2M preparation isadministered prior to the administration of the treatment modality. Inan alternate embodiment, the α2M preparation is administered subsequentto the administration of the treatment modality. In one embodiment, theα2M preparation is administered prior to the administration of thetreatment modality. In an alternate embodiment, the HSP preparation isadministered subsequent to the administration of the treatment modality.

In certain embodiments, the HSP preparation or the α2M preparation andnon-vaccine treatment modality are cyclically administered to a subject.Cycling therapy involves the administration of the HSP preparation for aperiod of time, followed by the administration of a treatment modalityfor a period of time and repeating this sequential administration.Alternatively, cycling therapy can involve the administration of α2Mpreparation for a period of time, followed by the administration of atreatment modality for a period of time and repeating this sequentialadministration. Cycling therapy can reduce the development of resistanceto one or more of the therapies, avoid or reduce the side effects of oneof the therapies, and/or improve the efficacy of the treatment. In suchembodiments, the invention contemplates the alternating administrationof an HSP preparation followed by the administration of a treatmentmodality 4 to 6 days later, preferable 2 to 4 days, later, morepreferably 1 to 2 days later, wherein such a cycle may be repeated asmany times as desired. The invention also contemplates the alternatingadministration of an α2M preparation followed by the administration of atreatment modality 4 to 6 days later, preferable 2 to 4 days, later,more preferably 1 to 2 days later, wherein such a cycle may be repeatedas many times as desired.

In certain embodiments, the HSP preparation and treatment modality arealternately administered in a cycle of less than 3 weeks, once every twoweeks, once every 10 days or once every week. In other embodiments, theα2M preparation and treatment modality are alternately administered incycles of less than 3 weeks, once every two weeks, once every 10 days oronce every week. In a specific embodiment of the invention, one cyclecan comprise the administration of a chemotherapeutic by infusion over90 minutes every cycle, 1 hour every cycle, or 45 minutes every cycle.Each cycle can comprise at least 1 week of rest, at least 2 weeks ofrest, at least 3 weeks of rest. In an embodiment, the number of cyclesadministered is from 1 to 12 cycles, more typically from 2 to 10 cycles,and more typically from 2 to 8 cycles.

In a preferred embodiment, an HSP preparation displaying theantigenicity of a tumor specific or tumor associated antigen of a typeof cancer is administered to a subject in an amount ineffective fortreating said cancer about 2 weeks to 1 month prior to receivingcombination chemotherapy with cytokine treatment, wherein treatmenteffectiveness is greater than the effectiveness of HSP preparation orcombination chemotherapy with cytokine treatment administered alone.Preferably the subject is human. In a preferred embodiment, the subjectis non-responsive to combination chemotherapy with cytokine treatmentprior to administration of HSP preparation. In another preferredembodiment, the chemotherapy is cyclophosphamide, the cytokine is IL-12,and the HSP preparation comprises gp96-peptide complexes obtained fromcancerous tissue of the subject.

In particularly preferred embodiment, an α2M preparation displaying theantigenicity of a tumor specific or tumor associated type of cancer isadministered to a subject in an amount ineffective for treating saidcancer about 2 weeks to 1 month prior to receiving combinationchemotherapy with cytokine treatment, wherein treatment effectiveness isgreater than the effectiveness of α2M preparation or combinationchemotherapy with cytokine treatment administered alone. Preferably thesubject is human. In a preferred embodiment, the subject isnon-responsive to combination chemotherapy with cytokine treatment priorto administration of α2M preparation. In another preferred embodiment,the chemotherapy is cyclophosphamide, the cytokine is IL-12, and the α2Mpreparation comprises α2M-peptide complexes obtained from canceroustissue of the subject.

In specific embodiments, the above methods encompass the administrationof Gleevec™ (imatinib mesylate) for treatment of cancer. In a preferredembodiment, the cancer is CML, the chemotherapeutic is Gleevec™(imatinib mesylate), and the HSP preparation comprises hsp70-peptidecomplexes obtained from the cancer subject being treated.

Also encompassed by the invention are methods of treatment and delivery,pharmaceutical compositions and formulas comprising administering atleast one non-vaccine therapeutic modality and an HSP preparation or anα2M preparation and kits comprising such pharmaceutical compositions.

5.2. Heat Shock Protein Preparations

Three major families of HSPs have been identified based on molecularweight. The families have been called hsp60, hsp70 and hsp90 where thenumbers reflect the approximate molecular weight of the stress proteinsin kilodaltons. Many members of these families were found subsequentlyto be induced in response to other stressful stimuli including, but notlimited to, nutrient deprivation, metabolic disruption, oxygen radicalsand infection with intracellular pathogens (See Welch, May 1993,Scientific American 56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420;Craig, 1993, Science 260:1902-1903; Gething, et al., 1992, Nature355:33-45; and Lindquist, et al., 1988, Annu. Rev. Genetics 22:631-677).A number of proteins thought to be involved in chaperoning functions areresidents of the endoplasmic reticulum (ER) lumen and include, forexample, protein disulfide isomerase (PDI; Gething et al., 1992, Nature355:33-45), calreticulin (Herbert et al., 1997, J. Cell Biol.139:613-623), Grp94 or ERp99 (Sorger & Pelham, 1987, J. Mol. Biol.194:(2) 341-4) which is related to hsp90, and Grp78 or BiP, which isrelated to hsp70 (Munro et al., 1986, Cell 46:291-300; Haas & Webl,1983, Nature 306:387-389). It is contemplated that HSPs belonging to allof these three families, including fragments of such HSPs, can be usedin the practice of the instant invention. It is also noted that HSPsinclude constitutively expressed conserved cellular homologs of theproteins induced by stress.

HSPs are also referred to interchangeably herein as stress proteins andcan be selected from among any cellular protein that satisfies thefollowing criteria. It is a protein whose intracellular concentrationincreases when a cell is exposed to a stressful stimuli, it is capableof binding other proteins or peptides, it is capable of releasing thebound proteins or peptides in the presence of adenosine triphosphate(ATP) or low pH, and it is a protein showing at least 35% homology withany cellular protein having any of the above properties.

Heat shock proteins are among the most highly conserved proteins inexistence. For example, DnaK, the hsp70 from E. coli has about 50% aminoacid sequence identity with hsp70 proteins from excoriates (Bardwell, etal., 1984, Proc. Natl. Acad. Sci. 81:848-852). The hsp60 and hsp90families also show similarly high levels of intra families conservation(Hickey, et al., 1989, Mol. Cell. Biol. 9:2615-2626; Jindal, 1989, Mol.Cell. Biol. 9:2279-2283). In addition, it has been discovered that thehsp60, hsp70 and hsp90 families are composed of proteins that arerelated to the stress proteins in sequence, for example, having greaterthan 35% amino acid identity, but whose expression levels are notaltered by stress. Therefore it is contemplated that stressproteins/HSPs include other proteins, muteins, analogs, and variantsthereof having at least 35% to 55%, preferably 55% to 75%, and mostpreferably 75% to 85% amino acid identity with members of the threefamilies whose expression levels in a cell are enhanced in response to astressful stimulus. The purification of stress proteins belonging tothese three families is described below.

In addition, HSPs have been found to have immunological and antigenicproperties. HSPs are now understood to play an essential role in immuneregulation. For instance, prior experiments have demonstrated that HSPsstimulate strong and long-lasting specific immune responses againstantigenic peptides that have been covalently or noncovalently attachedto the HSPs. By utilizing a specific peptide, the immune responsegenerated is “specific” or targeted to that peptide.

Where HSP-peptide complexes are used in conjunction with administrationof a non-vaccine treatment modality, preferably, the peptides areantigenic or relevant to the condition. In particular preferredembodiments, it is contemplated that the therapeutic outcome of atreatment modality administered to a subject with a particular type ofcancer is improved by the administration of an HSP-peptide complexwherein the peptide displays the antigenicity of an antigen of that typeof cancer.

In the present invention, an HSP preparation can include but not belimited to unbound hsp70, hsp90, gp96, calreticulin, hsp110 or grp170 ornoncovalent or covalent complexes thereof complexed to a peptide.

5.3. Preparation of Heat Shock Proteins and α2M

In the present invention, purified unbound HSPs, HSPs covalently ornoncovalently bound to specific peptides or nonspecific peptides(collectively referred to herein as HSP-peptide complexes), andcombinations of thereof are used. Purification of HSPs in complexed ornon-complexed forms are described in the following subsections. Further,one skilled in the art can synthesize HSPs by recombinant expression orpeptide synthesis, which are also described below.

Also encompassed by the present invention are purified unbound α2M, α2Mcovalently or noncovalently bound to specific peptides or nonspecificpeptides (collectively referred to herein as α2M-peptide complexes), andcombinations of thereof are used. Purification of α2M in complexed ornon-complexed forms are described in the following subsections. Further,one skilled in the art can synthesize α2M by recombinant expression orpeptide synthesis, which are also described below.

5.3.1. Preparation and Purification of Hsp70 or Hsp70-Peptide Complexes

The purification of noncovalently bound cellularly producedhsp70-peptide complexes has been described previously, see, for example,Udono et al., 1993, J. Exp. Med. 178:1391-1396. A procedure that may beused, presented by way of example but not limitation, is as follows:

Initially, human or mammalian cells are suspended in 3 volumes of 1×Lysis buffer consisting of 5 mM sodium phosphate buffer (pH 7), 150 mMNaCl, 2 mM CaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride(PMSF). Then, the pellet is sonicated, on ice, until>99% cells are lysedas determined by microscopic examination. As an alternative tosonication, the cells may be lysed by mechanical shearing and in thisapproach the cells typically are resuspended in 30 mM sodium bicarbonate(pH 7.5), 1 mM PMSF, incubated on ice for 20 minutes and thenhomogenized in a Dounce homogenizer until>95% cells are lysed.

Then the lysate is centrifuged at 1,000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose™ equilibratedwith phosphate buffered saline (PBS) containing 2 mM Ca²⁺ and 2 mM Mg²⁺.When the cells are lysed by mechanical shearing the supernatant isdiluted with an equal volume of 2× lysis buffer prior to mixing with ConA Sepharose™. The supernatant is then allowed to bind to the Con ASepharose™ for 2-3 hours at 4° C. The material that fails to bind isharvested and dialyzed for 36 hours (three times, 100 volumes each time)against 10 mM Tris-Acetate (pH 7.5), 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF.Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for20 minutes. Then the resulting supernatant is harvested and applied to aMono Q FPLC™ ion exchange chromatographic column (Pharmacia)equilibrated in 20 mM Tris-Acetate (pH 7.5), 20 mM NaCl, 0.1 mM EDTA and15 mM 2-mercaptoethanol. The column is then developed with a 20 mM to500 mM NaCl gradient and then eluted fractions fractionated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andcharacterized by immunoblotting using an appropriate anti-hsp70 antibody(such as from clone N27F3-4, from StressGen).

Fractions strongly immunoreactive with the anti-hsp70 antibody arepooled and the hsp70-peptide complexes precipitated with ammoniumsulfate; specifically with a 50%-70% ammonium sulfate cut. The resultingprecipitate is then harvested by centrifugation at 17,000 rpm (SS34Sorvall rotor) and washed with 70% ammonium sulfate. The washedprecipitate is then solubilized and any residual ammonium sulfateremoved by gel filtration on a Sephadex® G25 column (Pharmacia). Ifnecessary the hsp70 preparation thus obtained can be repurified throughthe Mono Q FPLC™ ion exchange chromatographic column (Pharmacia) asdescribed above.

The hsp70-peptide complex can be purified to apparent homogeneity usingthis method. Typically 1 mg of hsp70-peptide complex can be purifiedfrom 1 g of cells/tissue.

An improved method for purification of hsp70-peptide complexes comprisescontacting cellular proteins with ADP or a nonhydrolyzable analog of ATPaffixed to a solid substrate, such that hsp70 in the lysate can bind tothe ADP or nonhydrolyzable ATP analog, and eluting the bound hsp70. Apreferred method uses column chromatography with ADP affixed to a solidsubstratum (e.g., ADP-agarose). The resulting hsp70 preparations arehigher in purity and devoid of contaminating peptides. The hsp70 complexyields are also increased significantly by about more than 10 fold.Alternatively, chromatography with nonhydrolyzable analogs of ATP,instead of ADP, can be used for purification of hsp70-peptide complexes.By way of example but not limitation, purification of hsp70-peptidecomplexes by ADP-agarose chromatography can be carried out as follows:

Meth A sarcoma cells (500 million cells) are homogenized in hypotonicbuffer and the lysate is centrifuged at 100,000 g for 90 minutes at 4°C. The supernatant is applied to an ADP-agarose column. The column iswashed in buffer and is eluted with 5 column volumes of 3 mM ADP. Thehsp70-peptide complexes elute in fractions 2 through 10 of the total 15fractions which elute. The eluted fractions are analyzed by SDS-PAGE.The hsp70-peptide complexes can be purified to apparent homogeneityusing this procedure.

Separation of the HSP from an hsp70-peptide complex can be performed inthe presence of ATP or low pH. These two methods may be used to elutethe peptide from an hsp70-peptide complex. The first approach involvesincubating an hsp70-peptide complex preparation in the presence of ATP.The other approach involves incubating an hsp70-peptide complexpreparation in a low pH buffer. These methods and any others known inthe art may be applied to separate the HSP and peptide from anhsp-peptide complex.

5.3.2. Preparation and Purification of Hsp90 or Noncovalent CellularlyProduced Hsp90-Peptide Complexes

A procedure that can be used, presented by way of example and notlimitation, is as follows:

Initially, human or mammalian cells are suspended in 3 volumes of 1×Lysis buffer consisting of 5 mM sodium phosphate buffer (pH 7), 150 mMNaCl, 2 mM CaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride(PMSF). Then, the pellet is sonicated, on ice, until>99% cells are lysedas determined by microscopic examination. As an alternative tosonication, the cells may be lysed by mechanical shearing and in thisapproach the cells typically are resuspended in 30 mM sodium bicarbonate(pH 7.5), 1 mM PMSF, incubated on ice for 20 minutes and thenhomogenized in a Dounce homogenizer until>95% cells are lysed.

Then the lysate is centrifuged at 1,000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose™ equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cells are lysed bymechanical shearing the supernatant is diluted with an equal volume of2× Lysis buffer prior to mixing with Con A Sepharose™. The supernatantis then allowed to bind to the Con A Sepharose™ for 2-3 hours at 4° C.The material that fails to bind is harvested and dialyzed for 36 hours(three times, 100 volumes each time) against 10 mM Tris-Acetate (pH7.5), 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate iscentrifuged at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then theresulting supernatant is harvested and applied to a Mono Q FPLC™ ionexchange chromatographic column (Pharmacia) equilibrated with lysisbuffer. The proteins are then eluted with a salt gradient of 200 mM to600 mM NaCl.

The eluted fractions are fractionated by SDS-PAGE and fractionscontaining the hsp90-peptide complexes identified by immunoblottingusing an anti-hsp90 antibody such as 3G3 (Affinity Bioreagents).Hsp90-peptide complexes can be purified to apparent homogeneity usingthis procedure. Typically, 150-200 μg of hsp90-peptide complex can bepurified from 1 g of cells/tissue.

Separation of the HSP from an hsp90-peptide complex can be performed inthe presence of ATP or low pH. These two methods may be used to elutethe peptide from an hsp90-peptide complex. The first approach involvesincubating an hsp90-peptide complex preparation in the presence of ATP.The other approach involves incubating an hsp90-peptide complexpreparation in a low pH buffer. These methods and any others known inthe art may be applied to separate the HSP and peptide from anhsp-peptide complex.

5.3.3. Preparation and Purification of Gp96 or Noncovalent CellularlyProduced Gp96-Peptide Complexes

A procedure that can be used, presented by way of example and notlimitation, is as follows:

A pellet of human or mammalian cells is resuspended in 3 volumes ofbuffer consisting of 30 mM sodium bicarbonate buffer (pH 7.5) and 1 mMPMSF and the cells allowed to swell on ice 20 minutes. The cell pelletis then homogenized in a Dounce homogenizer (the appropriate clearanceof the homogenizer will vary according to each cell type) on iceuntil>95% cells are lysed.

The lysate is centrifuged at 1,000 g for 10 minutes to remove unbrokencells, nuclei and other debris. The supernatant from this centrifugationstep is then recentrifuged at 100,000 g for 90 minutes. The gp96-peptidecomplex can be purified either from the 100,000 pellet or from thesupernatant.

When purified from the supernatant, the supernatant is diluted withequal volume of 2× lysis buffer and the supernatant mixed for 2-3 hoursat 4° C. with Con A Sepharose™ equilibrated with PBS containing 2 mMCa²⁺ and 2 mM Mg²⁺. Then, the slurry is packed into a column and washedwith 1× lysis buffer until the OD₂₈₀ drops to baseline. Then, the columnis washed with 1/3 column bed volume of 10% α-methyl mannoside (α-MM)dissolved in PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺, the column sealedwith a piece of parafilm, and incubated at 37° C. for 15 minutes. Thenthe column is cooled to room temperature and the parafilm removed fromthe bottom of the column. Five column volumes of the α-MM buffer areapplied to the column and the eluate analyzed by SDS-PAGE. Typically theresulting material is about 60-95% pure, however this depends upon thecell type and the tissue-to-lysis buffer ratio used. Then the sample isapplied to a Mono Q FPLC™ ion exchange chromatographic column(Pharmacia) equilibrated with a buffer containing 5 mM sodium phosphate(pH 7). The proteins are then eluted from the column with a 0-1M NaClgradient and the gp96 fraction elutes between 400 mM and 550 mM NaCl.

The procedure, however, may be modified by two additional steps, usedeither alone or in combination, to consistently produce apparentlyhomogeneous gp96-peptide complexes. One optional step involves anammonium sulfate precipitation prior to the Con A purification step andthe other optional step involves DEAE-Sepharose™ purification after theCon A purification step but before the Mono Q FPLC™ step.

In the first optional step, described by way of example as follows, thesupernatant resulting from the 100,000 g centrifugation step is broughtto a final concentration of 50% ammonium sulfate by the addition ofammonium sulfate. The ammonium sulfate is added slowly while gentlystirring the solution in a beaker placed in a tray of ice water. Thesolution is stirred from about ½ to 12 hours at 4° C. and the resultingsolution centrifuged at 6,000 rpm (Sorvall SS34 rotor). The supernatantresulting from this step is removed, brought to 70% ammonium sulfatesaturation by the addition of ammonium sulfate solution, and centrifugedat 6,000 rpm (Sorvall SS34 rotor). The resulting pellet from this stepis harvested and suspended in PBS containing 70% ammonium sulfate inorder to rinse the pellet. This mixture is centrifuged at 6,000 rpm(Sorvall SS34 rotor) and the pellet dissolved in PBS containing 2 mMCa²⁺ and Mg²⁺. Undissolved material is removed by a brief centrifugationat 15,000 rpm (Sorvall SS34 rotor). Then, the solution is mixed with ConA Sepharose™ and the procedure followed as before.

In the second optional step, described by way of example as follows, thegp96 containing fractions eluted from the Con A column are pooled andthe buffer exchanged for 5 mM sodium phosphate buffer (pH 7), 300 mMNaCl by dialysis, or preferably by buffer exchange on a Sephadex G25column. After buffer exchange, the solution is mixed withDEAE-Sepharose™ previously equilibrated with 5 mM sodium phosphatebuffer (pH 7), 300 mM NaCl. The protein solution and the beads are mixedgently for 1 hour and poured into a column. Then, the column is washedwith 5 mM sodium phosphate buffer (pH 7), 300 mM NaCl, until theabsorbance at 280 nm drops to baseline. Then, the bound protein iseluted from the column with five volumes of 5 mM sodium phosphate buffer(pH 7), 700 mM NaCl. Protein containing fractions are pooled and dilutedwith 5 mM sodium phosphate buffer (pH 7) in order to lower the saltconcentration to 175 mM. The resulting material then is applied to theMono Q FPLC™ ion exchange chromatographic column (Pharmacia)equilibrated with 5 mM sodium phosphate buffer (pH 7) and the proteinthat binds to the Mono Q FPLC™ ion exchange chromatographic column(Pharmacia) is eluted as described before.

It is appreciated, however, that one skilled in the art may assess, byroutine experimentation, the benefit of incorporating the secondoptional step into the purification protocol. In addition, it isappreciated also that the benefit of adding each of the optional stepswill depend upon the source of the starting material.

When the gp96 fraction is isolated from the 100,000 g pellet, the pelletis suspended in 5 volumes of PBS containing either 1% sodiumdeoxycholate or 1% oxtyl glucopyranoside (but without the Mg²⁺ and Ca²⁺)and incubated on ice for 1 hour. The suspension is centrifuged at 20,000g for 30 minutes and the resulting supernatant dialyzed against severalchanges of PBS (also without the Mg²⁺ and Ca²⁺) to remove the detergent.The dialysate is centrifuged at 100,000 g for 90 minutes, thesupernatant harvested, and calcium and magnesium are added to thesupernatant to give final concentrations of 2 mM, respectively. Then thesample is purified by either the unmodified or the modified method forisolating gp96-peptide complex from the 100,000 g supernatant, seeabove.

The gp96-peptide complexes can be purified to apparent homogeneity usingthis procedure. About 10-20 μg of gp96 can be isolated from 1 gcells/tissue.

Separation of the HSP from an gp96-peptide complex can be performed inthe presence of ATP or low pH. These two methods may be used to elutethe peptide from an gp96-peptide complex. The first approach involvesincubating an gp96-peptide complex preparation in the presence of ATP.The other approach involves incubating an gp96-peptide complexpreparation in a low pH buffer. These methods and any others known inthe art may be applied to separate the HSP and peptide from anhsp-peptide complex.

5.3.4. Preparation and Purification of Noncovalent Cellularly ProducedHsp110-Peptide Complexes

A procedure, described by Wang et al., 2001, J. Immunol. 166(1):490-7,that can be used, presented by way of example and not limitation, is asfollows:

A pellet (40-60 ml) of cell or tissue, e.g., tumor cell tissue, ishomogenized in 5 vol of hypotonic buffer (30 mN sodium bicarbonate,pH7.2, and protease inhibitors) by Dounce homogenization. The lysate iscentrifuged at 4,500×g and then 100,000×g for 2 hours. If the cells ortissues are of hepatic origin, the resulting supernatant is was firstapplied to a blue Sepharose column (Pharmacia) to remove albumin.Otherwise, the resulting supernatant is applied to a Con A-Sepharosecolumn (Pharmacia Biotech, Piscataway, N.J.) previously equilibratedwith binding buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM MgCl₂; 1mM CaCl₂; 1 mM MnCl₂; and 15 mM 2-ME). The bound proteins are elutedwith binding buffer containing 15% α-D-o-methylmannoside (Sigma, St.Louis, Mo.).

Con A-Sepharose unbound material is first dialyzed against a solution of20 mM Tris-HCl, pH 7.5; 100 mM NaCl; and 15 mM 2-ME, and then applied toa DEAE-Sepharose column and eluted by salt gradient from 100 to 500 mMNaCl. Fractions containing hsp110 are collected, dialyzed, and loadedonto a Mono Q (Pharmacia) 10/10 column equilibrated with 20 mM Tris-HCl,pH 7.5; 200 mM NaCl; and 15 mM 2-ME. The bound proteins are eluted witha 200-500 mM NaCl gradient. Fractions are analyzed by SDS-PAGE followedby immunoblotting with an Ab for hsp110, as described by Wang et al.,1999, J. Immunol. 162:3378. Pooled fractions containing hsp110 areconcentrated by Centriplus (Amicon, Beverly, Mass.) and applied to aSuperose 12 column (Pharmacia). Proteins are eluted by 40 mM Tris-HCl,pH 8.0; 150 mM NaCl; and 15 mM 2-ME with a flow rate of 0.2 ml/min.

5.3.5. Preparation and Purification of Noncovalent Cellularly ProducedGrp170-Peptide Complexes

A procedure, described by Wang et al., 2001, J. Immunol. 166(1):490-7,that can be used, presented by way of example and not limitation, is asfollows:

A pellet (40-60 ml) of cell or tissue, e.g., tumor cell tissue, ishomogenized in 5 vol of hypotonic buffer (30 mN sodium bicarbonate,pH7.2, and protease inhibitors) by Dounce homogenization. The lysate iscentrifuged at 4,500×g and then 100,000×g for 2 hours. If the cells ortissues are of hepatic origin, the resulting supernatant is was firstapplied to a blue Sepharose column (Pharmacia) to remove albumin.Otherwise, the resulting supernatant is applied to a Con A-Sepharosecolumn (Pharmacia Biotech, Piscataway, N.J.) previously equilibratedwith binding buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM MgCl₂; 1mM CaCl₂; 1 mM MnCl₂; and 15 mM 2-ME). The bound proteins are elutedwith binding buffer containing 15% α-D-o-methylmannoside (Sigma, St.Louis, Mo.).

Con A-Sepharose-bound material is first dialyzed against 20 mM Tris-HCl,pH 7.5, and 150 mind NaCl and then applied to a Mono Q column and elutedby a 150 to 400 mM NaCl gradient. Pooled fractions are concentrated andapplied on the Superose 12 column (Pharmacia). Fractions containinghomogeneous grp170 are collected.

5.3.6. α2M-Antigenic Molecule Complexes

Endogenous α2M-antigenic molecule complexes can be obtained by thefollowing non-limiting methods.

Alpha-2-macroglobulin can be bought from commercial sources or preparedby purifying it from human blood. To purify α2M from blood, thefollowing non-limiting protocol can be used:

Blood is collected from a subject and is allowed to clot. It is thencentrifuged for 30 minutes under 14,000×g to obtain the serum which isthen applied to a gel filtration column (Sephacryl S-300R) equilibratedwith 0.04M Tris buffer pH 7.6 plus 0.3M NaCl. A 65 ml column is used forabout 10 ml of serum. Three ml fractions are collected and each fractionis tested for the presence of α2M by dot blot using an α2M specificantibody. The α2M positive fractions are pooled and applied to a PD10column to exchange the buffer to 0.01M Sodium Phosphate buffer pH 7.5with PMSF. The pooled fractions are then applied to a Con A column (10ml) equilbrated with the phosphate buffer. The column is washed and theprotein is eluted with 5% methylmannose pyranoside. The eluent is passedover a PD10 column to change the buffer to a Sodium Acetate buffer(0.05M; pH6.0). A DEAE column is then equilibrated with acetate bufferand the sample is applied to the DEAE column. The column is washed andthe protein is eluted with 0.13M sodium acetate. The fractions with α2Mare then pooled.

5.3.6. Recombinant Expression of HSPs and α2M and Antigenic Peptides

Methods known in the art can be utilized to recombinantly produce HSPsand α2M. A nucleic acid sequence encoding a heat shock protein orencoding α2M can be inserted into an expression vector for propagationand expression in host cells.

An expression construct, as used herein, refers to a nucleotide sequenceencoding an HSP or α2M operably associated with one or more regulatoryregions which enables expression of the HSP or α2M in an appropriatehost cell. “Operably-associated” refers to an association in which theregulatory regions and the HSP or α2M sequence to be expressed arejoined and positioned in such a way as to permit transcription, andultimately, translation.

The regulatory regions necessary for transcription of the HSP or α2M canbe provided by the expression vector. A translation initiation codon(ATG) may also be provided if the HSP or α2M gene sequence lacking itscognate initiation codon is to be expressed. In a compatiblehost-construct system, cellular transcriptional factors, such as RNApolymerase, will bind to the regulatory regions on the expressionconstruct to effect transcription of the modified HSP or α2M sequence inthe host organism. The precise nature of the regulatory regions neededfor gene expression may vary from host cell to host cell. Generally, apromoter is required which is capable of binding RNA polymerase andpromoting the transcription of an operably-associated nucleic acidsequence. Such regulatory regions may include those 5′ non-codingsequences involved with initiation of transcription and translation,such as the TATA box, capping sequence, CAAT sequence, and the like. Thenon-coding region 3′ to the coding sequence may contain transcriptionaltermination regulatory sequences, such as terminators andpolyadenylation sites.

In order to attach DNA sequences with regulatory functions, such aspromoters, to the HSP or α2M gene sequence or to insert the HSP or α2Mgene sequence into the cloning site of a vector, linkers or adaptersproviding the appropriate compatible restriction sites may be ligated tothe ends of the cDNAs by techniques well known in the art (Wu et al.,1987, Methods in Enzymol 152:343-349). Cleavage with a restrictionenzyme can be followed by modification to create blunt ends by digestingback or filling in single-stranded DNA termini before ligation.Alternatively, a desired restriction enzyme site can be introduced intoa fragment of DNA by amplification of the DNA by use of PCR with primerscontaining the desired restriction enzyme site.

An expression construct comprising an HSP or α2M sequence operablyassociated with regulatory regions can be directly introduced intoappropriate host cells for expression and production of HSP-peptidecomplexes and α2M-peptide complexes without further cloning. See, forexample, U.S. Pat. No. 5,580,859. The expression constructs can alsocontain DNA sequences that facilitate integration of the HSP or α2Msequence into the genome of the host cell, e.g., via homologousrecombination. In this instance, it is not necessary to employ anexpression vector comprising a replication origin suitable forappropriate host cells in order to propagate and express the HSP or α2Min the host cells.

A variety of expression vectors may be used including, but not limitedto, plasmids, cosmids, phage, phagemids or modified viruses. Typically,such expression vectors comprise a functional origin of replication forpropagation of the vector in an appropriate host cell, one or morerestriction endonuclease sites for insertion of the HSP or α2M genesequence, and one or more selection markers. The expression vector mustbe used with a compatible host cell which may be derived from aprokaryotic or an eukaryotic organism including but not limited tobacteria, yeasts, insects, mammals and humans.

For long term, high yield production of properly processed HSP/α2M orHSP-peptide/α2M-peptide complexes, stable expression in mammalian cellsis preferred. Cell lines that stably express HSP/α2M orHSP-peptide/α2M-peptide complexes may be engineered by using a vectorthat contains a selectable marker. By way of example but not limitation,following the introduction of the expression constructs, engineeredcells may be allowed to grow for 1-2 days in an enriched media, and thenare switched to a selective media. The selectable marker in theexpression construct confers resistance to the selection and optimallyallows cells to stably integrate the expression construct into theirchromosomes and to grow in culture and to be expanded into cell lines.Such cells can be cultured for a long period of time while HS/α2M P isexpressed continuously.

The recombinant cells may be cultured under standard conditions oftemperature, incubation time, optical density and media composition.However, conditions for growth of recombinant cells may be differentfrom those for expression of HSPs/α2M and antigenic proteins. Modifiedculture conditions and media may also be used to enhance production ofthe HSP/α2M. For example, recombinant cells containing HSPs with theircognate promoters may be exposed to heat or other environmental stress,or chemical stress. Any techniques known in the art may be applied toestablish the optimal conditions for producing HSP/α2M orHSP-peptide/α2M-peptide complexes.

Cells may be derived from a variety of sources, including, but notlimited to, cells infected with an infectious agent and cancer cells andinclude, but are not limited to, epithelial cells, endothelial cells,keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells suchas T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc. The choice of cell type depends on the type of tumor orinfectious disease being treated or prevented, and can be determined byone of skill in the art. In a specific embodiment, an expressionconstruct comprising a nucleic acid sequence encoding the HSP/α2Mpolypeptide is introduced into an antigenic cell. As used herein,antigenic cells may include cells that are infected with an infectiousagent or pathogen, cells infected with non-infectious or non-pathogenicforms of an infectious agent or pathogen (e.g., by use of a helperinfectious agent), cells infected by or engineered to express anattenuated form of an infectious agent or a non-pathogenic orreplication-deficient variant of a pathogen, pre-neoplastic cells thatare infected with a cancer-causing infectious agent, such as a virus,but which are not yet neoplastic; or antigenic cells that have beenexposed to a mutagen or cancer-causing agent, such as, for exampleDNA-damaging agents, radiation, etc. Other cells that can be used arepre-neoplastic cells which are in transition from a normal to aneoplastic form as characterized by morphology, physiological orbiochemical functions. Preferably, the cancer cells and pre-neoplasticcells used in the methods of the invention are of mammalian origin.Mammals contemplated by this aspect of the invention include humans,companion animals (e.g., dogs and cats), livestock animals (e.g., sheep,cattle, goats, pigs and horses), laboratory animals (e.g., mice, ratsand rabbits), and captive or free wild animals.

In various embodiments, any cancer cell, preferably a human cancer cell,can be used in the present methods for producing the peptide-complexes.The cancer cells provide the antigenic peptides which become associatedcovalently or noncovalently with the expressed HSP/α2M polypeptide. Thepeptide-complexes are then purified from the cells and used to treatsuch cancers. Cancers which can be treated or prevented with immunogeniccompositions prepared by methods of the invention include, but are notlimited to, tumors such as sarcomas and carcinomas. Accordingly, anytissues or cells isolated from a pre-neoplastic lesion, a cancer,including cancer that has metastasized to multiple remote sites, can beused in the present method. For example, cells found in abnormallygrowing tissue, circulating leukemic cells, metastatic lesions as wellas solid tumor tissue can be used.

In another embodiment, cell lines derived from a pre-neoplastic lesion,cancer tissues or cancer cells can also be used, provided that the cellsof the cell line have at least one or more antigenic determinants incommon with antigens on the target cancer cells. Cancer tissues, cancercells, cells infected with a cancer-causing agent, other pre-neoplasticcells, and cell lines of human origin are preferred.

Cancer and pre-neoplastic cells can be identified by any method known inthe art. For example, cancer cells can be identified by morphology,enzyme assays, proliferation assays, cytogenetic characterization, DNAmapping, DNA sequencing, the presence of cancer-causing virus, or ahistory of exposure to mutagen or cancer-causing agent, imaging, etc.Cancer cells may also be obtained by surgery, endoscopy, or other biopsytechniques. If some distinctive characteristics of the cancer cells areknown, they can also be obtained or purified by any biochemical orimmunological methods known in the art, such as but not limited toaffinity chromatography, and fluorescence activated cell sorting (e.g.,with fluorescently tagged antibody against an antigen expressed by thecancer cells).

Cancer tissues, cancer cells or cell lines may be obtained from a singleindividual or pooled from several individuals. It is not essential thatclonal, homogeneous, or purified population of cancer cells be used. Itis also not necessary to use cells of the ultimate target in vivo (e.g.,cells from the tumor of the intended recipient), so long as at least oneor more antigenic determinants on the target cancer cells is present onthe cells used for expression of the HSP/α2M polypeptide. In addition,cells derived from distant metastases may be used to prepare animmunogenic composition against the primary cancer. A mixture of cellscan be used provided that a substantial number of cells in the mixtureare cancer cells and share at least one antigenic determinant with thetarget cancer cell. In a specific embodiment, the cancer cells to beused in expressing an HSP/α2M polypeptide are purified.

5.3.5. Peptide Synthesis

An alternative to producing HSP/α2M by recombinant techniques is peptidesynthesis. For example, an entire HSP/α2M, or a peptide corresponding toa portion of an HSP/α2M can be synthesized by use of a peptidesynthesizer. Conventional peptide synthesis or other synthetic protocolswell known in the art may be used.

Peptides having the amino acid sequence of an HSP/α2M or a portionthereof may be synthesized by solid-phase peptide synthesis usingprocedures similar to those described by Merrifield, 1963, J. Am. Chem.Soc., 85:2149, During synthesis, N-α-protected amino acids havingprotected side chains are added stepwise to a growing polypeptide chainlinked by its C-terminal and to an insoluble polymeric support i.e.,polystyrene beads. The peptides are synthesized by linking an aminogroup of an N-α-deprotected amino acid to an α-carboxyl group of anN-α-protected amino acid that has been activated by reacting it with areagent such as dicyclohexylcarbodiimide. The attachment of a free aminogroup to the activated carboxyl leads to peptide bond formation. Themost commonly used N-α-protecting groups include Boc which is acidlabile and Fmoc which is base labile. Details of appropriatechemistries, resins, protecting groups, protected amino acids andreagents are well known in the art and so are not discussed in detailherein (See, Atherton, et al., 1989, Solid Phase Peptide Synthesis: APractical Approach, IRL. Press, and Bodanszky, 1993, Peptide Chemistry,A Practical Textbook, 2nd Ed., Springer-Verlag).

Purification of the resulting HSP/α2M is accomplished using conventionalprocedures, such as preparative HPLC using gel permeation, partitionand/or ion exchange chromatography. The choice of appropriate matricesand buffers are well known in the art and so are not described in detailherein.

5.4. Antigenic Molecules

The following subsections provide an overview of peptides that areuseful as antigenic/immunogenic components of the HSP/α2M-peptidecomplexes of the invention, and how such peptides can be identified,e.g., for use in recombinant expression of the peptides for in vitrocomplexing of HSPs/α2M and antigenic molecules. However, in the practiceof the present invention, the identity of the antigenic molecule(s) ofthe HSP/α2M peptide-complex need not be known, for example when theHSP/α2M complex is purified directly from a cancerous cell or from atissue infected with a pathogen.

5.4.1. Isolation of Antigenic/Immunogenic Components

It has been found that antigenic peptides and/or components can beeluted from HSP/α2M complexes either in the presence of ATP or low pH.These experimental conditions may be used to isolate peptides and/orantigenic components from cells which may contain potentially usefulantigenic determinants. Once isolated, the amino acid sequence of eachantigenic peptide may be determined using conventional amino acidsequencing methodologies. Such antigenic molecules can then be producedby chemical synthesis or recombinant methods, purified, and complexed toHSPs in vitro to form the HSP complexes of the invention.

Similarly, it has been found that potentially immunogenic peptides maybe eluted from MHC-peptide complexes using techniques well known in theart (Falk, K. et al., 1990 Nature 348:248-251; Elliott, T., et al.,1990, Nature 348:195-197; Falk, K., et al., 1991, Nature 351:290-296).

Thus, potentially immunogenic or antigenic peptides may be isolated fromeither endogenous stress protein-peptide complexes or endogenousMHC-peptide complexes for use subsequently as antigenic molecules, bycomplexing in vitro to HSP/α2M to form the HSP/α2M complexes of theinvention. Exemplary protocols for isolating peptides and/or antigeniccomponents from either of these complexes are known in the art aredescribed hereinbelow.

5.4.2. Peptides From Stress Protein-Peptide Complexes

Two methods may be used to elute the peptide from a stressprotein-peptide complex.

One approach involves incubating the stress protein-peptide complex inthe presence of ATP. The other approach involves incubating thecomplexes in a low pH buffer.

Briefly, the complex of interest is centrifuged through a Centricon 10assembly (Millipore) to remove any low molecular weight material looselyassociated with the complex. The large molecular weight fraction may beremoved and analyzed by SDS-PAGE while the low molecular weight may beanalyzed by HPLC as described below. In the ATP incubation protocol, thestress protein-peptide complex in the large molecular weight fraction isincubated with 10 mM ATP for 30 minutes at room temperature. In the lowpH protocol, acetic acid or trifluoroacetic acid (TFA) is added to thestress protein-peptide complex to give a final concentration of 10%(vol/vol) and the mixture incubated at room temperature or in a boilingwater bath or any temperature in between, for 10 minutes (See, VanBleek, et al., 1990, Nature 348:213-216; and Li, et al., 1993, EMBOJournal 12:3143-3151).

The resulting samples are centrifuged through a Centricon 10 assembly asmentioned previously. The high and low molecular weight fractions arerecovered. The remaining large molecular weight stress protein-peptidecomplexes can be reincubated with ATP or low pH to remove any remainingpeptides.

The resulting lower molecular weight fractions are pooled, concentratedby evaporation and dissolved in 0.1% TFA. The dissolved material is thenfractionated by reverse phase high pressure liquid chromatography (HPLC)using for example a VYDAC C18 reverse phase column equilibrated with0.1% TFA. The bound material is then eluted at a flow rate of about 0.8ml/min by developing the column with a linear gradient of 0 to 80%acetonitrile in 0.1% TFA. The elution of the peptides can be monitoredby OD210 and the fractions containing the peptides collected.

5.4.3. Peptides from MHC-Peptide Complexes

The isolation of potentially immunogenic peptides from MHC molecules iswell known in the art and so is not described in detail herein (See,Falk, et al., 1990, Nature 348:248-251; Rotzsche, et al., 1990, Nature348:252-254; Elliott, et al., 1990, Nature 348:191-197; Falk, et al.,1991, Nature 351:290-296; Demotz, et al., 1989, Nature 343:682-684;Rotzsche, et al., 1990, Science 249:283-287), the disclosures of whichare incorporated herein by reference.

Briefly, MHC-peptide complexes may be isolated by a conventionalimmunoaffinity procedure. The peptides then may be eluted from theMHC-peptide complex by incubating the complexes in the presence of about0.1% TFA in acetonitrile. The eluted peptides may be fractionated andpurified by reverse phase HPLC, as before.

The amino acid sequences of the eluted peptides may be determined eitherby manual or automated amino acid sequencing techniques well known inthe art. Once the amino acid sequence of a potentially protectivepeptide has been determined the peptide may be synthesized in anydesired amount using conventional peptide synthesis or other protocolswell known in the art.

Peptides having the same amino acid sequence as those isolated above maybe synthesized by solid-phase peptide synthesis using procedures similarto those described by Merrifield, 1963, Am. Chem. Soc., 85:2149. Duringsynthesis, N-α-protected amino acids having protected side chains areadded stepwise to a growing polypeptide chain linked by its C-terminaland to an insoluble polymeric support i.e., polystyrene beads. Thepeptides are synthesized by linking an amino group of an N-α-deprotectedamino acid to an α-carboxy group of an N-α-protected amino acid that hasbeen activated by reacting it with a reagent such asdicyclohexylcarbodiimide. The attachment of a free amino group to theactivated carboxyl leads to peptide bond formation. The most commonlyused N-α-protecting groups include Boc which is acid labile and Fmocwhich is base labile.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe polystyrene beads. The N-α-protecting group is then removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-α-protected amino acid. The process is repeateduntil the desired peptide is synthesized. The resulting peptides arethen cleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides can be derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein (See, Atherton, etal., 1989, Solid Phase Peptide Synthesis; A Practical Approach, IRLPress, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2ndEd., Springer-Verlag).

Purification of the resulting peptides is accomplished usingconventional procedures, such as preparative HPLC using gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

5.4.4. Exogenous Antigenic Molecules

Molecules that display the antigenicity of a known antigen of a pathogenor of a tumor-specific or tumor-associated antigen of a cancer type,e.g. antigens or antigenic portions thereof, can be selected for use asantigenic molecules, for complexing to HSP/α2M, from among those knownin the art or determined by immunoassay to be able to bind to antibodyor MHC molecules (antigenicity) or generate immune response(immunogenicity). To determine immunogenicity or antigenicity bydetecting binding to antibody, various immunoassays known in the art canbe used, including but not limited to competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitin reactions, immunodiffusion assays, invivo immunoassays (using colloidal gold, enzyme or radioisotope labels,for example), western blots, immunoprecipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labelled.Many means are known in the art for detecting binding in an immunoassayand are envisioned for use. In one embodiment for detectingimmunogenicity, T cell-mediated responses can be assayed by standardmethods, e.g., in vitro cytoxicity assays or in vivo delayed-typehypersensitivity assays.

Potentially useful antigens or derivatives thereof for use as antigenicmolecules can also be identified by various criteria, such as theantigen's involvement in neutralization of a pathogen's infectivity(wherein it is desired to treat or prevent infection by such a pathogen)(Norrby, 1985, Summary, in Vaccines 85, Lerner, et al. (eds.), ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 388-389), typeor group specificity, recognition by patients' antisera or immune cells,and/or the demonstration of protective effects of antisera or immunecells specific for the antigen. In addition, where it is desired totreat or prevent a disease caused by pathogen, the antigen's encodedepitope should preferably display a small or no degree of antigenicvariation in time or amongst different isolates of the same pathogen.

Preferably, where it is desired to treat or prevent cancer, knowntumor-specific (i.e., expressed in tumor cells) or tumor associatedantigens (i.e., relatively overexpressed in tumor cells) or fragments orderivatives thereof are used. For example, such tumor specific ortumor-associated antigens include but are not limited to KS 1/4pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol.142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415); ovarian carcinomaantigen (CA125) (Yu, et al, 1991, Cancer Res. 51(2):468-475); prostaticacid phosphate (Miler, et al., 1990, Nucl. Acids Res. 18(16):4928);prostate specific antigen (Henttu and Vihko, 1989, Biochem. Biophys.Res. Comm. 160(2):903-910; Israeli, et al., 1993, Cancer Res.53:227-230); melanoma-associated antigen p97 (Estin, et al., 1989, J.Natl. Cancer Inst. 81(6):445-446); melanoma antigen gp75 (Vijayasardahl,et al., 1990, J. Exp. Med. 171(4):1375-1380); high molecular weightmelanoma antigen (Natali, et al., 1987, Cancer 59:55-63) and prostatespecific membrane antigen. Other exogenous antigens that may becomplexed to HSPs/α2M include portions or proteins that are mutated at ahigh frequency in cancer cells, such as oncogenes (e.g., ras, inparticular mutants of ras with activating mutations, which only occur infour amino acid residues (12, 13, 59 or 61) (Gedde-Dahl et al., 1994,Eur. J. Immunol. 24(2):410-414)) and tumor suppressor genes (e.g., p53,for which a variety of mutant or polymorphic p53 peptide antigenscapable of stimulating a cytotoxic T cell response have been identified(Gnjatic et al., 1995, Eur. J. Immunol. 25(6):1638-1642).

In a specific embodiment, an antigen or fragment or derivative thereofspecific to a certain tumor is selected for complexing to HSPs/α2M toform an HSP/α2M complex for administration to a patient having thattumor.

Preferably, where it is desired to treat or prevent viral diseases,molecules comprising epitopes of known viruses are used. For example,such antigenic epitopes may be prepared from viruses including, but notlimited to, hepatitis type A, hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II). Preferably, where it is desired to treat or preventbacterial infections, molecules comprising epitopes of known bacteriaare used. For example, such antigenic epitopes may be prepared frombacteria including, but not limited to, mycobacteria rickettsia,mycoplasma, neisseria and legionella.

Preferably, where it is desired to treat or prevent protozoalinfections, molecules comprising epitopes of known protozoa are used.For example, such antigenic epitopes may be prepared from protozoaincluding, but not limited to, leishmania, kokzidioa, and trypanosoma.

Preferably, where it is desired to treat or prevent parasiticinfections, molecules comprising epitopes of known parasites are used.For example, such antigenic epitopes may be from parasites including,but not limited to, chlamydia and rickettsia.

5.5. In Vitro Production of Non-Covalent HSP/α2M Complexes

In an embodiment in which HSPs/α2M and the peptides with which they areendogenously associated in vivo are not employed, complexes of HSPs/α2Mto antigenic molecules are produced in vitro. As will be appreciated bythose skilled in the art, the peptides either isolated by theaforementioned procedures or chemically synthesized or recombinantlyproduced may be reconstituted with a variety of purified natural orrecombinant stress proteins in vitro to generate immunogenicnon-covalent stress protein-antigenic molecule complexes. Alternatively,exogenous antigens or antigenic or immunogenic fragments or derivativesthereof can be complexed to stress proteins. A preferred, exemplaryprotocol for complexing a stress protein and an antigenic molecule invitro is discussed below.

In a method which produces non-covalent HSP-antigenic molecule complexesand α2M-antigenic molecule complexes, a complex is prepared according tothe method described by Blachere et al., 1997 J. Exp. Med.186(8):1315-22, which incorporated by reference herein in its entirety.Blachere teaches in vitro complexing of hsps to antigenic molecule. Theprotocol described in Blachere can be modified such that the hspcomponent is substituted by α2M. Binder et at. (2001, J. Immunol.166:4968-72) demonstrates that the Blachere method yields complexes ofα2M bound to antigenic molecules.

Prior to complexing, the HSPs/α2M are pretreated with ATP or low pH toremove any peptides that may be associated with the HSP/α2M of interest.When the ATP procedure is used, excess ATP is removed from thepreparation by the addition of apyranase as described by Levy, et al.,1991, Cell 67:265-274, When the low pH procedure is used, the buffer isreadjusted to neutral pH by the addition of pH modifying reagents.

The antigenic molecules and the pretreated HSP/α2M are admixed to givean approximately 5 antigenic molecule: 1 stress protein molar ratio.Then, the mixture is incubated for 15 minutes to 3 hours at 4° to 45° C.in a suitable binding buffer such as one containing 20 mM sodiumphosphate, pH 7.2, 350 mM NaCl, 3 mM MgCl2 and 1 mM phenyl methylsulfonyl fluoride (PMSF). The preparations are centrifuged through aCentricon 10 assembly (Millipore) to remove any unbound peptide. Theassociation of the peptides with the stress proteins can be assayed bySDS-PAGE, This is the preferred method for in vitro complexing ofpeptides isolated from MHC-peptide complexes of peptides disassociatedfrom endogenous HSP peptide complexes.

In an alternative embodiment of the invention, preferred for producingcomplexes of hsp70 to exogenous antigenic molecules such as proteins,5-10 micrograms of purified HSP is incubated with equimolar quantitiesof the antigenic molecule in 20 mM sodium phosphate buffer pH 7.5, 0,5MNaCl, 3 mM MgCl2 and 1 mM ADP in a volume of 100 microliter at 37° C.for 1 hr. This incubation mixture is further diluted to 1 ml inphosphate-buffered saline.

In an alternative embodiment of the invention, preferred for producingcomplexes of gp96 or hsp90 to peptides, 5-10 micrograms of purified gp96or hsp90 is incubated with equimolar or excess quantities of theantigenic peptide in a suitable buffer such as one containing 20 mMsodium phosphate buffer pH 7.5, 0.5M NaCl, 3 nM MgCl2 at 60-65° C. for5-20 min. This incubation mixture is allowed to cool to room temperatureand centrifuged one or more times if necessary, through a Centricon 10assembly (Millipore) to remove any unbound peptide.

Antigenic molecules may be isolated from various sources, chemicallysynthesized, or produced recombinantly. Such methods can be readilyadapted for medium or large scale production of the immunotherapeutic orprophylactic vaccines.

Following complexing, the immunogenic antigenic molecule complexes canoptionally be assayed in vitro using, for example, the mixed lymphocytetarget cell assay (MLTC) described below. Once immunogenic complexeshave been isolated they can be optionally characterized further inanimal models using the preferred administration protocols andexcipients discussed below.

5.6. Formation of Covalent HSP/α2M Complexes

As an alternative to non-covalent complexes of HSPs/α2M and antigenicmolecules, antigenic molecules may be covalently attached to HSPs/α2M.HSP/α2M peptide complexes are preferably cross-linked after theirpurification from cells or tissues. Covalently linked complexes are thecomplexes of choice when a B cell response is desired.

In one embodiment, HSPs/α2M are covalently coupled to antigenicmolecules by chemical crosslinking. Chemical crosslinking methods arewell known in the art. For example, in a preferred embodiment,glutaraldehyde crosslinking may be used. Glutaradehyde crosslinking hasbeen used for formation of covalent complexes of peptides and hsps (seeBarrios et al., 1992, Eur. J. Immunol. 22: 1365-1372). Preferably, 1-2mg of HSP peptide complex is crosslinked in the presence of 0.002%glutaraldehyde for 2 hours. Glutaraldehyde is removed by dialysisagainst phosphate buffered saline (PBS) overnight (Lussow et al., 1991,Eur. J. Immunol. 21: 2297-2302). In one embodiment, the followingprotocol is used. Optionally, HSPs may be pretreated with ATP or low pHprior to complexing, in order to remove any peptides that may beassociated with the HSP polypeptide. Preferably, 1 mg of HSP iscrosslinked to 1 mg of peptide in the presence of 0.002% glutaraldehydefor 2 hours. Glutaraldehyde is removed by dialysis against phosphatebuffered saline (PBS) overnight (Lussow et al., 1991, Eur. J. Immunol.21: 2297-2302).

Other methods for chemical crosslinking may also be used, in additionother methods for covalent attachment of proteins, such asphotocrosslinking (see Current Protocols in Molecular Biology, Ausubelet al. (eds.), Greene Publishing Associates and Wiley Interscience, NewYork).

In another embodiment, the HSP and specific antigen(s) are crosslinkedby ultraviolet (UV) crosslinking.

In one embodiment, HSPs are covalently coupled to peptide fragments bychemical crosslinking. Chemical crosslinking methods are well known inthe art. For example, in a preferred embodiment, glutaraldehydecrosslinking may be used. Glutaradehyde crosslinking has been used forformation of covalent complexes of peptides and HSPs (see Barrios etal., 1992, Eur. J. Immunol. 22: 1365-1372). Preferably, 1-2 mg ofHSP-peptide complex is crosslinked in the presence of 0.002%glutaraldehyde for 2 hours. Glutaraldehyde is removed by dialysisagainst phosphate buffered saline (PBS) overnight (Lussow et al., 1991,Eur. J. Immunol. 21: 2297-2302). Alternatively, an HSP and a populationof peptides can be crosslinked by ultraviolet (UV) crosslinking underconditions known in the art.

In another embodiment of the invention, a population of peptides can becomplexed to α2M by incubating the peptide fragments with α2M at a 50:1molar ratio and incubated at 50° C. for 10 minutes followed by a 30minute incubation at 25° C. Free (uncomplexed) peptides are then removedby size exclusion filters. Protein-peptide complexes are preferablymeasured by a scintillation counter to make sure that on a per molarbasis, each protein is observed to bind equivalent amounts of peptide(approximately 0.1% of the starting amount of the peptide). For details,see Binder, 2001, J. Immunol. 166(8):4968-72, which is incorporatedherein by reference in its entirety.

Alternatively, a population of antigenic peptides can be complexed toα2M covalently by methods as described in PCT publications WO 94/14976and WO 99/50303 for complexing a peptide to α2M, which are incorporatedherein by reference in their entirety. Covalent linking of a populationof antigenic peptides to α2M can be performed using a bifunctionalcrosslinking agent. Such crosslinking agents and methods of their useare also well known in the art.

In general, when an α2M is mixed with a protease, cleavage of the “bait”region of α2M takes place, the proteinase becomes “trapped” bythioesters, and a conformational change takes place that allows bindingof the α2M complex to the α2M receptor. During proteolytic activation ofα2M, non-proteolytic ligands can become covalently bound to theactivated thioesters. Non-proteolytic ligands can also be incorporatedinto the activated α2M molecule by ammonia or methylamine duringreversal of the nucleophilic activation, employing heat (Gram and Pizzo,1998, Biochemistry, 37: 6009-6014). Such conditions that allowfortuitous trapping of peptides by α2M are employed to prepare theα2M-antigenic complexes for use in the invention. Methods for suchcovalent coupling have been described previously (Osada et al., 1987,Biochem. Biophys. Res. Commun. 146:26-31; Osada et al., 1988, Biochem.Biophys. Res. Commun. 150:883; Chu and Pizzo, 1993, J. Immunol. 150:48;Chu et al., 1994, Ann. N.Y. Acad. Sci. 737:291-307; Mitsuda et al.,1993, Biochem. Biophys. Res. Commun. 101:1326-1331). Thus in oneembodiment, an α2M antigenic molecule complex can be prepared asdescribed by Grøn and Pizzo, 1998, Biochemistry, 37: 6009-6014. Themethod of Grøn and Pizzo yields complexes of α2M that are covalentlybound to antigenic molecules.

For example, α2M polypeptide is mixed with an antigenic molecule in thepresence of a protease, ammonia or other small amine nucleophiles suchas methylamine and ethylamine. Non-limiting examples of proteases whichmay be used include trypsin, porcine pancreatic elastase (PEP), humanneutrophil elastase, cathepsin G, S. aureus V-8 proteinase trypsin,α-chymotrypsin, V8 protease, papain, and proteinase K (see Ausubel etal., eds., in “Current Protocols in Molecular Biology”, GreenePublishing Associates and Wiley Interscience, New York, 17.4.6-17.4.8).A preferred, exemplary protocol for complexing an α2M polypeptide and anantigenic molecule in vitro follows. The antigenic molecules (1 μg-20mg) and the α2M polypeptide (1 μg-20 mg) are mixed together inphosphate-buffered saline (PBS) (100 μl-5 ml) in the presence of aprotease, such as trypsin (0.92 mg trypsin in approximately 500 μl PBS,to give an approximately 5:1 antigenic molecule:α2M polypeptide molarratio. The mixture is then incubated for 5-15 minutes at 37° C. 500 μl 4mg/ml p-Aphenyl methyl sulfonyl fluoride (p-APMSF) is added to thesolution to inhibit trypsin activity and incubated for 2 hrs at 25° C.The preparations can be centrifuged through a Centricon 10 assembly(Millipore) to remove any unbound peptide. Alternatively, free antigenicmolecule may be removed by passage over a gel permeation column. Theassociation of the peptides with the α2M polypeptide can be assayed bySDS-PAGE. This is the preferred method for in vitro complexing ofantigenic molecules isolated from MHC-antigenic molecule complexes, orpeptides disassociated from endogenous α2M-antigenic molecule complexes.The foregoing methods could readily be used to generate HSP-peptidecomplexes.

5.7. HSP or α2M Fusion Proteins

In certain embodiments of the invention, an HSP/α2M antigenic moleculecomplex is a recombinant fusion protein. Such recombinant fusionproteins, comprised of HSP/α2 μM sequences linked to antigenic moleculesequences, may be used in the methods of the present invention. Toproduce such a recombinant fusion protein, an expression vector isconstructed using nucleic acid sequences encoding the HSP/α2M fused tosequences encoding an antigenic molecule, using recombinant methodsknown in the art (see Suzue et al., 1997, Proc. Natl. Acad. Sci. U.S.A.94: 13146-51). HSP/α2M antigenic peptide fusions are then expressed andisolated. By specifically designing the antigenic peptide portion of themolecule, such fusion proteins can be used to elicit an immune responseand in immunotherapy against target cancer and infectious diseases.

5.8. Kits, Dosage Regimens, Administration and Formulations

Kits are also provided for carrying out the therapeutic methods of thepresent invention. In one embodiment, a kit comprises a first containercontaining a purified HSP preparation or α2M preparation and a secondcontainer containing a non-vaccine therapeutic modality for treatment ofcancer. Preferably, the cancer is CML, the HSP preparation compriseshsp70-peptide complexes, and the therapeutic modality is Gleevec™. In aspecific embodiment, the second container contains imatinib mesylate. Inanother specific embodiment, the imatinib mesylate is purified. In aspecific embodiment, a kit comprises a first container containing apurified HSP preparation or α2M preparation in an amount ineffective totreat a disease or disorder when administered alone; and a secondcontainer containing a non-vaccine treatment modality in an amount that,when administered before, concurrently with, or after the administrationof the HSP preparation or α2M preparation in the first container, iseffective to improve overall treatment effectiveness over theeffectiveness of the administration of each component alone. In anotherspecific embodiment, a kit comprises a first container containing apurified HSP preparation or α2M preparation in an amount ineffective totreat a disease or disorder when administered alone; and a secondcontainer containing one or more non-vaccine treatment modalities in anamount that, when administered before, concurrently with, or after theadministration of the HSP preparation or α2M preparation in the firstcontainer, is effective to improve overall treatment effectiveness overthe effectiveness of the administration of the HSP preparation or α2Mpreparation administered alone or the treatment modalities administeredalone. In yet another specific embodiment, a first container containinga purified HSP preparation or α2M preparation in an amount ineffectiveto treat a disease or disorder when administered alone; and a secondcontainer and third container, each containing a non-vaccine treatmentmodality in an amount that, when administered before, concurrently with,or after the administration of the HSP preparation or α2M preparation inthe first container, is effective to improve overall treatmenteffectiveness over the effectiveness of the administration of HSPpreparation or α2M preparation administered alone or treatmentmodalities administered alone. In a preferred specific embodiment, theinvention provides a kit comprising in a first container, a purified HSPpreparation or α2M comprising a population of noncovalent HSP-peptidecomplexes α2M-peptide complexes obtained from cancerous tissue of amammal; in a second container, a composition comprising a purifiedcancer chemotherapeutic agent; and in a third container, a compositioncomprising a purified cytokine. In a specific embodiment, the secondcontainer containing imatinib mesylate contains purified imatinibmesylate.

The dosage of HSP preparation or μ2 M preparation to be administereddepends to a large extent on the condition and size of the subject beingtreated as well as the amount of non-vaccine treatment modalityadministered, the frequency of treatment and the route ofadministration. Regimens for continuing therapy, including site, doseand frequency may be guided by the initial response and clinicaljudgment.

Depending on the route of administration and the type of HSPs in the HSPpreparation, the amount of HSP in the HSP preparation can range, forexample, from 0.1 to 1000 μg per administration. The preferred amountsof gp96 or hsp70 are in the range of 10 to 600 μg per administration and0.1 to 100 μg if the HSP preparation is administered intradermally. Aparticularly preferred amount of hsp70 is about 50 μg per administrationif administered intradermally. For hsp 90, the preferred amounts areabout 50 to 1000 μg per administration, and about 5 to 50 μg forintradermal administration. The amount of α2M administered can rangefrom 2 to 1000 μg, preferably 20 to 500 μg, most preferably about 25 to250 μg, given once weekly for about 4-6 weeks, intradermally with thesite of administration varied sequentially.

Because in certain embodiments, the methods of the invention useadministration of HSP preparation in sub-optimal amounts, it isenvisioned that depending on the route of administration and the type ofHSPs in the HSP preparation, the amount of HSP in the HSP preparationcan be less than an amount in the range of 0.1 to 1000 μg peradministration. Accordingly, the preferred amounts of gp96 or hsp70 arein amounts less than the range of 10 to 600 μg per administration andless than the range of 0.1 to 10 μg if the HSP preparation isadministered intradermally. For hsp 90, the preferred amounts are lessthan the range of 50 to 1000 μg per administration, and less than therange of 5 to 50 μg for intradermal administration. The amount of α2Madministered can range from less than the range of 2 to 1000 μg,preferably less than the range of 20 to 500 μg, most preferably lessthan the range of 25 to 250 μg, given once weekly for about 4-6 weeks,intradermally with the site of administration varied sequentially.

Solubility and the site of the administration of the treatment modalityare factors which should be considered when choosing the route ofadministration of the HSP preparation of the invention. The mode ofadministration can be varied, including, but not limited to, e.g.,subcutaneously, intravenously, intraperitoneally, intramuscularly,intradermally or mucosally. Mucosal routes can further take the form oforal, rectal and nasal administration. With the above factors taken intoaccount, it may be preferable to administer the HSP to a site that isthe same or proximal to the site of administration of the treatmentmodality.

In an embodiment of the invention, HSPs/α2M may be administered usingany desired route of administration. Advantages of intradermaladministration include use of lower doses and rapid absorption,respectively. Advantages of subcutaneous or intramuscular administrationinclude suitability for some insoluble suspensions and oily suspensions,respectively. Mucosal routes of administration include, but are notlimited to, oral, rectal and nasal administration. Preparations formucosal administrations are suitable in various formulations asdescribed below.

If the HSP/α2M preparation is water-soluble, then it may be formulatedin an appropriate buffer, for example, phosphate buffered saline orother physiologically compatible solutions, preferably sterile.Alternatively, if the resulting complex has poor solubility in aqueoussolvents, then it may be formulated with a non-ionic surfactant such asTween, or polyethylene glycol. Thus, the compounds and theirphysiologically acceptable solvates may be formulated for administrationby inhalation or insufflation (either through the mouth or the nose) ororal, buccal, parenteral, or rectal administration or, in the case oftumors, directly injected into a solid tumor.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such a liquid preparation may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalpreparation may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

The HSP/α2M preparation for oral administration may be suitablyformulated to give controlled release of the active compound.

For buccal administration, the preparation may take the form of tabletsor lozenges formulated in conventional manner.

The preparation may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The preparationmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The preparation may also be formulated in a rectal preparation such as asuppository or retention enema, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the preparationmay also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the preparation may be formulated with suitable polymericor hydrophobic materials (for example, as an emulsion in an acceptableoil) or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt. Liposomes and emulsions are wellknown examples of delivery vehicles or carriers for hydrophilic drugs.

For administration by inhalation, the preparation for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The preparation may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theHSP preparation or α2M preparation. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

The appropriate and recommended dosages, formulation and routes ofadministration for treatment modalities such as chemotherapeutic agents,radiation therapy and biological/immunotherapeutic agents such ascytokines are known in the art and described in such literature as thePhysician's Desk Reference (56^(th) ed., 2002). In particularembodiments, the present invention comprises administering ananti-cancer agent such as any of those described below in Table 2,preferably for the treatment of breast, ovary, melanoma, prostate, colonor lung cancer, CML or soft tissue sarcomas, including but not limitedto gastrointestinal stromal tumors as described below in section 5.11.

Because in certain embodiments, the methods of the invention compriseadministration of sub-optimal amounts of the therapeutic modality, it isenvisioned that the dosages of each therapeutic modality can be lessthan that used in standard therapy or known in the art.

In one embodiment, Gleevec™ is administered 50 mg to 100 mg, 100 mg to200 mg, 200 mg to 300 mg, 300 mg to 400 mg, 400 mg to 500 mg, 500 mg to600 mg, 600 mg to 700 mg, 700 mg to 800 mg, 800 mg to 900 mg, or 900 mgto 1000 mg daily. In certain embodiments, the total daily dose isadministered to a subject as two daily doses of 25 mg to 50 mg, 50 mg to100 mg, 100 mg to 200 mg, 200 mg to 300 mg, 300 mg to 400 mg, or 400 mgto 500 mg. Gleevec™ is administered orally in dosages of 100 mg to 1000mg, preferably 200 mg to 900 mg, more preferably 300 mg to 800 mg, mostpreferably 400 mg to 600 mg. In a specific embodiment, Gleevec™ isadministered orally, at a sub-optimal daily dosage. In preferredembodiments, the sub-optimal daily dosage of orally administeredGleevec™ is about 10 mg to 600 mg, about 50 mg to 400 mg, about 100 mgto 300 mg, or about 200 mg. In other embodiments, Gleevec™ isadministered orally every other day, every third day, every fourth day,every fifth day, every sixth day, or once a week, at a dosage of 100 mgto 800 mg, 200 mg to 600 mg, 300 mg to 500 mg, or 400 mg.

TABLE 2 Therapeutic Agent Dose/Administration/Formulation imatinibmesylate Oral 400-600 mg daily (Gleevec ™) (capsule) Capsules eachcontain imatinib mesylate equivalent to 100 mg imatinib free basedoxorubicin Intravenous 60-75 mg/m² on Day 1 21 day intervalshydrochloride (Adriamycin RDF ® and Adriamycin PFS ®) epirubicinIntravenous 100-120 mg/m² on Day 1 of each 3-4 week cycles hydrochloridecycle or (Ellence ™) divided equally and given on Days 1-8 of the cyclefluorousacil Intravenous How supplied: 5 mL and 10 mL vials (containing250 and 500 mg flourouracil respectively) docetaxel Intravenous 60-100mg/m² over 1 hour Once every 3 weeks (Taxotere ®) paclitaxel Intravenous175 mg/m² over 3 hours Every 3 weeks for (Taxol ®) 4 courses(administered sequentially to doxorubicin- containing combinationchemotherapy) tamoxifen citrate Oral 20-40 mg Daily (Nolvadex ®)(tablet) Dosages greater than 20 mg should be given in divided doses(morning and evening) leucovorin calcium Intravenous or How supplied:Dosage is unclear from text. for injection intramuscular 350 mg vial PDR3610 injection luprolide acetate Single 1 mg (0.2 mL or 20 unit mark)Once a day (Lupron ®) subcutaneous injection flutamide Oral (capsule)250 mg 3 times a day at 8 hour (Eulexin ®) (capsules contain 125 mgintervals (total daily dosage flutamide each) 750 mg) nilutamide Oral300 mg or 150 mg 300 mg once a day for 30 (Nilandron ®) (tablet)(tablets each contain 50 or 150 days followed by 150 mg mg nilutamide)once a day bicalutamide Oral 50 mg Once a day (Casodex ®) (tablet)(tablets each contain 50 mg bicalutamide) progesterone Injection USP insesame oil 50 mg/mL ketoconazole Cream 2% cream applied once or twice(Nizoral ®) daily depending on symptoms prednisone Oral Initial dosagemay vary from 5 (tablet) mg to 60 mg per day depending on the specificdisease entity being treated estramustine Oral 14 mg/kg of body weight(i.e. Daily given in 3 or 4 divided phosphate sodium (capsule) one 140mg capsule for each 10 doses (Emcyt ®) kg or 22 lb of body weight)etoposide or Intravenous 5 mL of 20 mg/mL solution VP-16 (100 mg)dacarbazine Intravenous 2-4.5 mg/kg Once a day for 10 days.(DTIC-Dome ®) May be repeated at 4 week intervals polifeprosan 20 waferplaced 8 wafers, each containing 7.7 mg with carmustine in resection ofcarmustine, for a total of 61.6 implant (BCNU) cavity mg, if size andshape of (nitrosourea) resection cavity allows (Gliadel ®) cisplatinInjection [n/a in PDR 861] How supplied: solution of 1 mg/mL in multi-dose vials of 50 mL and 100 mL mitomycin Injection supplied in 5 mg and20 mg vials (containing 5 mg and 20 mg mitomycin) gemcitabine HClIntravenous For NSCLC-2 schedules have 4 week schedule- (Gemzar ®) beeninvestigated and the Days 1, 8 and 15 of each 28- optimum schedule hasnot been day cycle. Cisplatin determined intravenously at 100 mg/m² 4week schedule- on day 1 after the infusion of administrationintravenously at Gemzar. 1000 mg/m² over 30 minutes on 3 week schedule-3 week schedule- Days 1 and 8 of each 21 day Gemzar administered cycle.Cisplatin at dosage of intravenously at 1250 mg/m² 100 mg/m²administered over 30 minutes intravenously after administration ofGemzar on day 1. carboplatin Intravenous Single agent therapy: Every 4weeks (Paraplatin ®) 360 mg/m² I.V. on day 1 (infusion lasting 15minutes or longer) Other dosage calculations: Combination therapy withcyclophosphamide, Dose adjustment recommendations, Formula dosing, etc.ifosamide Intravenous 1.2 g/m² daily 5 consecutive days (Ifex ®) Repeatevery 3 weeks or after recovery from hematologic toxicity topotecanIntravenous 1.5 mg/m² by intravenous 5 consecutive days, startinghydrochloride infusion over 30 minutes daily on day 1 of 21 day course(Hycamtin ®)

5.10. Treatment and Prevention of Infectious Diseases

Infectious diseases that can be treated using the methods of the presentinvention are caused by infectious agents including, but not limited to,viruses, bacteria, fungi protozoa and parasites.

Infectious agents that can be treated according to the inventioninclude, but are not limited to viruses, bacteria, fungi, and agents ofprotozoal disease.

Viral diseases that can be treated or prevented using the methods of thepresent invention include, but are not limited to, those caused byhepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-1), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, small pox,Epstein Barr virus, human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), and agents of viral diseasessuch as viral meningitis, encephalitis, dengue or small pox.

Bacterial diseases that can be treated or prevented by use of themethods of the present invention are caused by bacteria including, butnot limited to, mycobacteria rickettsia, mycoplasma, neisseria, S.pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus antracis(anthrax), tetanus, streptococcus, staphylococcus, mycobacterium,tetanus, pertissus, cholera, plague, diptheria, chlamydia, S. aureus andlegionella.

Protozoal diseases that can be treated or prevented by use of animmunoreactive reagent in conjunction with the methods of the presentinvention are caused by protozoa including, but not limited to,leishmania, kokzidioa, trypanosoma or malaria.

Parasitic diseases that can be treated or prevented by use of themethods of the present invention are caused by parasites including, butnot limited to, chlamydia and rickettsia.

5.11. Treatment of Cancer

A number of non-vaccine cancer treatment modalities are currently inclinical trials and well-known in the art. The HSP/α2M preparation canbe used in conjunction with such non-vaccine cancer treatment modalitiesfor the treatment and prevention of the respective types of cancers. Oneskilled in the art would be able to determine experimental and standardanti-cancer therapies and treatments that could be used according to themethods of the present invention.

Cancers that can be treated using the methods of the present inventioninclude, but are not limited to human sarcomas and carcinomas, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemias, acute lymphocytic leukemia, acute myelocyticleukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocyticleukemia, monocytic leukemia, erythroleukemia, chronic leukemia, chronicmyeloid leukemia, chronic myelogenous leukemia, chronic myelocyticleukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia,polycythemia vera, lymphoma, Hodgkin's disease lymphoma, non-Hodgkin'sdisease lymphoma, multiple myeloma, Waldenström's macroglobulinemia,heavy chain disease, soft tissue sarcomas, gastrointestinal stromaltumors, and glioblastomas.

6. EXAMPLE TUMORS NON-RESPONSIVE TO CHEMOTHERAPY/CYTOKINE TREATMENTRESPOND AFTER ADMINISTRATION OF HSP-PEPTIDE COMPLEX

Mice bearing tumors, such as LLC (D122) and B16, do not respond totreatment of Cyclophosphamide (Cy) in combination with interleukin-12(IL-12). In a double-graft experiment, mice were injected with MCA207(tumors known to respond to Cy+IL-12 treatment) and D122 at two oppositeflanks, and tumors were allowed to grow to a significant size (10×10mm), then the mice were treated with Cy+IL-12. The large MCA207 tumorsregressed rapidly, whereas the D122 tumors continued to grow on theopposite flank of the same animals. The results demonstrated thatcertain tumors, e.g., D122 do not respond to Cy+IL-12 treatment, eventhough a vigorous response against another tumor is present in the sameanimal.

The tumors that responded to the treatment appear to be immunogenic,whereas other tumors that are non-responders were all poorlyimmunogenic. To test whether a host-derived immune recognition of thetumor in the forms of T cell priming prior to Cy+IL-12 treatment wouldresult in the tumor responding to treatment, the following experimentswere conducted. The following results demonstrated that if a mousebearing a D 122 tumor that does not respond to Cy+IL-12 treatment aloneobtains immunological memory of the tumor, a tumor rejection will occurin the mice following treatment with Cy+IL-12.

Heat shock protein-peptide complexes were used for eliciting a robust Tcell response that includes both CD4+ and CD8+ T cells in mice.

6.1 Materials and Methods

Naïve mice were either un-immunized, or immunized once at day 0 with 5and 20 μg D 122-derived gp96-peptide complexes administeredsubcutaneously, or 2 μg D122-derived gp96-peptide complexes administeredintradermally. As a negative control, another group of mice wereimmunized with liver-derived gp96-peptide complexes. The D122-derivedgp96-peptide complexes are HSP-peptide complexes endogenous to andisolated from D122 tumor cells. The liver-derived gp96-peptide complexesare HSP-peptide complexes endogenous to and isolated form liver cells.Two weeks after the immunization (day 14), the mice were challengedsubcutaneously with 200,000 D122 cells. The immunization was sub-optimalfor tumor rejection according to our previous experience and D122 tumorsgrew in all mice. When tumor size reached 10 mm or above in diameter(day 32-34), the mice were treated with Cy+IL-12 (Cy, 3 mg byintraparenteral administration; IL-12, 200 ng, intraparenteraladministration for 5 days).

6.2. Results

Cure rate Size of tumors cured Mice immunized with (#/total) (mm indiameter) PBS 2/16  7 and 10 Liver-derived gp96-peptide complexes 2/1010 and 12 D122-derived gp96-peptide complexes 11/12  From 8 to 22

As summarized in the table above, upon antigen-specific immunologicalstimulation with autologous tumor derived gp96-peptide complexes,non-responder tumor D122 became a responder to the treatment ofCy+IL-12. In groups un-immunized and immunized with liver-derivedgp96-peptide complexes, only those mice bearing the smallest tumors(less than 10-12 mm in diameter) experienced tumor regression afterCy+IL-12 treatment. In contrast, in mice that were immunized with D122-derived gp96-peptide complexes, large D122 tumors such as those 22mm in diameter, which are generally refractory to any kind ofimmunotherapeutic approaches reported, regressed completely after theCy+IL-12 treatment. In addition, immunohistochemistry analysis for anumber of tumor samples harvested from each group reveals that 1) Nosign of T cell infiltration in the tumors removed from mice that wereun-immunized or immunized with liver-derived gp96-peptide complexesbefore or after the Cy+IL-12 treatment; 2) In contrast, some T cellinfiltration (both CD4+ and CD8+) was observed in tumors harvested frommice immunized with D122-derived gp96-peptide complexes 12 days, but not6 days, after the Cy+IL-12 treatment was initiated.

7. EXAMPLE COMPLETE ELIMINATION OF LEUKEMIA CELLS IN PATIENTS IN CHRONICPHASE CML AFTER ADMINISTRATION OF COMBINATION GLEEVEC™ AND HSP-PEPTIDECOMPLEX

To test the feasibility of immunization with autologous tumor-derivedhsp70-peptide complexes to treat patients in chronic phase CML, thefollowing protocol was used (FIG. 1). The clinical protocol summarizedin FIG. 1 includes all physical examinations, blood work, x-rays andbone marrows that were done before, during and after vaccination with anHSP preparation. Prior to inclusion in the study, subjects' diagnosis ofCML was confirmed by bcr/abl molecular typing of peripheral blood orbone marrow obtained from the subject using polymerase chain reaction(PCR) to determine the presence or absence of bcr/abl chimeric proteinsor transcripts.

7.2 Materials and Methods

Subjects that participated fulfilled the following criteria: subjectdisplayed an Eastern Cooperative Oncology Group (ECOG) performance scoreless than 2; subject was at least 18 years of age, and capable of givinginformed consent; less than one year has passed since the originaldiagnosis of Philadelphia chromosome positive CML in chronic phase;subject was not in cytogenetic remission; subject was not anticipating abone marrow or stem cell transplant within the next six months unlesssuch therapy was deemed necessary by a treatment physician due toevolution of the disease; subjects were allowed to maintain concurrentstandard treatment hydroxyurea, Ara-C/day for 10 days or Gleevec™(imatinib mesylate); subject lacked any serious illness such thatmedical condition might be compromised by participation in the study;subject showed adequate renal function as measured by serum creatininelevels less than 2.0, and adequate hepatic function, as measured bybilirubin and transaminase less than 2.0 times the upper normal limit;subject was not on corticosteroid therapy, or other immunosuppressivemedication; and subject did not display a lack of energy as shown byadequate delayed type hypersensitivity (DHT) response to at least 1 outof 3 antigens by skin testing with Candida, mumps and PPD, i.e.,induration was greater than 0.5 cm 48 hours after placement.

Subjects were excluded if: subject displayed an ECOG performancescore≧2; subject was more than 3 years out from original diagnosis ofPhiladelphia chromosome positive CML in chronic phase; subject was onIFN treatment; subject showed significant anemia, i.e., hemoglobin lessthan 10 g/dl or thrombocytopenia, i.e., platelet less than 20,000/μl,requiring transfusion; subject showed peripheral blast count over 10%;subject showed positive urine or blood pregnancy test; subject showedimpaired renal function, i.e., serum creatine greater than or equal to2.0, or impaired hepatic function, i.e., bilirubin or transaminase morethan 2.0 times the upper normal limit; subject showed significant activeinfection requiring hospitalization at time of enrollment; subject withsignificant behavioral or psychological problems that prevented adequatefollow-up.

A subject was discontinued for any of the following reasons: subjectrequested to withdraw for any reason; a proven effective therapeuticapproach became available, and was preferred by the subject (e.g., theapproval of other investigational medications by the regulatory agency,identification of an identical human leukocyte antigen (HLA) matcheddonor), the subject was lost to follow-up; the subject showed clearevidence of disease acceleration despite concurrent therapy as evidencedby the following signs and symptoms: peripheral blasts 10% or more;peripheral blast plus promyelocytes 30% or more; peripheral basophils20% or more; thrombocytopenia less than 100,000/mm³ unrelated totherapy; neutropenia less than 1,000/mm³ unrelated to therapy; marrowblast 10% or more; significant marrow fibrosis; progressive splenomaglyunresponsive to therapy; triad of WBC greater than 50,000/mm³,hematocrit less than 25% and platelets less than 100,000/mm³ notcontrolled with therapy; persistent unexplained fever; and cytogeneticclonal evolution; extramedullary disease with localized immature blastsuch as chloroma; any other reason which in the opinion of theinvestigator was to protect the best interest of the subject.

Prior to administration of their first HSP preparation, subjects hadbeen receiving Gleevec™ therapy (400-800 mg daily in capsule form,400-600 mg daily doses administered once a day, or 800 mg in two dailydoses of 400 mg each) for 2 days, 5 months, 9 months, 10 months, and 1year, respectively. Subjects satisfying the above criteria were allowedto remain on Gleevec™ therapy throughout the study. Subjectssubsequently underwent aphaeresis using peripheral vein access tocollect peripheral mononuclear cells. The majority of the specimen wasused for the purification of Hsp70-peptide complexes. The autologoushsp70-peptide complexes were then purified using an ADP-agaroseprotocol, substantially as described in Section 5.3.1 above. A smallfraction of the collection was used as targets for a CTL, assay.Subjects received an intradermal injection of 50 μg hsp70-peptidecomplexes in the skin of the forearm weekly over a two month period fora total of 8 injections, in addition to Gleevec™ therapy (400-800 mgdaily in capsule form, 400-600 mg daily doses administered once a day,800 mg doses administered twice a day). Blood samples were drawn threetimes to access the status of the immune system. Blood was collectedprior to the vaccination, during the vaccination and 1-2 weeks after the8^(th) vaccination (see FIG. 1). At the end of the treatment, allsubjects underwent full hematological and cytogenetic staging on thebone marrow (see Silver et al., 1999, Blood 94(5):1517-1536).

In addition, to collect feasibility and toxicity data, the developmentof anti-tumor immunity was measured according to methods known in theart, such as: (1) an increase in peripheral blood of IFN-γ producingCD8+ T-lymphocytes which are reactive to the autologous bcr/abl positiveperipheral mononuclear cells (see e.g., Janetzki et al., 2000, Int. J.Cancer 88:232-238); (2) an increase of PR-1 specific CTLs by PR1-HLA-A2tetramer techniques in patients who are HLA-A2 positive (see e.g., Clarket al., 2001, Blood 98(10): 2887-2893 and Molldrem et al., 1999, CancerResearch 59: 2675-2681); (3) the change of immunophenotype of peripherallymphocytes (see e.g., Akel et al., 2002, Clin. Lab. Haem. 24:362-367);and (4) the cytogenetic remission of Philadelphia chromosome from thebone marrow (see e.g., Wang at, 2002, British J. Haematology118:771-777).

Combined treatment in the five evaluable subjects resulted in completeelimination of leukemia cells as determined by: RT-PCR analysisdetermining the presence or absence of bcr/abl transcripts in theperipheral blood or bone marrow collected from treated patients, (seee.g., Merx et al., 2002, Leukemia 16:1579-1583; Wang et al., 2002,British Journal of Haematology 118: 771-777; and Stentoft et al, 2001,Eur. J. Haemotol. 67: 302-308); cytogenetic response, one of thecriteria used in the approval of Gleevec™ (see Silver et al, 1999, Blood94(5):1517-1536); or a combination of RT-PCR and cytogenetic response.Based on previous reports, less than 10 percent of patients treated withGleevec™ alone achieve responses using these same criteria. See Drukeret al., 2002, Hematology (Am. Soc. Hematol. Educ. Program): 111-135, at114-115.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. A method of treating a cancer in a subject inneed thereof comprising: administering to the subject a purified heatshock protein preparation comprising a heat shock protein-peptidecomplex comprising a heat shock protein covalently or noncovalentlyattached to an antigenic peptide, wherein the antigenic peptide displaysantigenicity of an antigen of the cancer, and wherein the subject hasbeen administered Bevacizumab to treat the cancer.
 2. The method ofclaim 1, wherein the purified heat shock protein preparation comprisesheat shock protein-peptide complexes that comprise one or more of hsp70,hsp90, hsp110, gp96, grp170, and calreticulin.
 3. The method of claim 2,wherein the purified heat shock protein preparation comprises heat shockprotein-peptide complexes that comprise gp96.
 4. The method of claim 1,wherein the subject receives further administrations of Bevacizumab andthe purified heat shock protein preparation, wherein the administrationsare repeated for up to 12 cycles.
 5. The method of claim 1, comprisingadministering the purified heat shock protein preparation once a weekfor the first 4 weeks.
 6. The method of claim 5, comprisingadministering the purified heat shock protein once every other weekafter the first 4 weeks of administration.
 7. The method of claim 1,wherein the purified heat shock protein preparation and the Bevacizumabare administered 2 to 4 days apart, 4 to 6 days apart, 1 week apart, 1to 2 weeks apart, or 2 to 4 weeks apart.
 8. The method of claim 1, whichfurther comprises the step of administering to the subject radiationtherapy.
 9. The method of claim 1, wherein the purified heat shockprotein preparation is administered for a first period of time, followedby administration of Bevacizumab for a second period of time, whereinthis sequential administration is repeated.
 10. The method of claim 1,wherein the Bevacizumab is administered prior to an initialadministration of the purified heat shock protein preparation.
 11. Themethod of claim 1, wherein Bevacizumab is administered concurrently withthe administration of the purified heat shock protein preparation. 12.The method of claim 1, wherein the Bevacizumab is administeredsubsequent to an initial administration of the purified heat shockprotein preparation.
 13. The method of claim 1, wherein the purifiedheat shock protein preparation is cyclically administered withBevacizumab.
 14. The method of claim 1, wherein the purified heat shockprotein preparation is autologous to the subject.
 15. The method ofclaim 1, wherein the subject is human.
 16. The method of claim 15,wherein the purified heat shock protein preparation comprises heat shockprotein-peptide complexes isolated from cells of the cancer of thesubject.
 17. The method of claim 1, wherein the purified heat shockprotein preparation comprises heat shock protein-peptide complexescomprising heat shock proteins non-covalently attached to antigenicpeptides.
 18. The method of claim 1, wherein the purified heat shockprotein preparation comprises heat shock protein-peptide complexesisolated from cells of the cancer.
 19. The method of claim 1, whereinthe antigenic peptide displays antigenicity of a tumor-specific antigenor a tumor-associated antigen of the cancer in the subject.
 20. Themethod of claim 1, wherein the cancer is a relapse form of cancer. 21.The method of claim 12, wherein the subsequent Bevacizumabadministration is administered no more than 24 hours after the initialadministration of the purified heat shock protein preparation.
 22. Themethod of claim 1, wherein the purified heat shock protein preparationis administered intradermally.
 23. The method of claim 22, wherein theBevacizumab is administered intravenously.
 24. A method of treating acancer in a subject in need thereof, wherein the subject is a candidatefor receiving Bevacizumab to treat the cancer, comprising: (a)administering to the subject Bevacizumab; and (b) administering to thesubject a purified heat shock protein preparation comprising a heatshock protein-peptide complex comprising a heat shock protein covalentlyor noncovalently attached to an antigenic peptide, wherein the antigenicpeptide displays antigenicity of an antigen of the cancer.
 25. Themethod of claim 24, wherein the purified heat shock protein preparationcomprises heat shock protein-peptide complexes that comprise one or moreof hsp70, hsp90, hsp110, gp96, grp170, and calreticulin.
 26. The methodof claim 25, wherein the purified heat shock protein preparationcomprises heat shock protein-peptide complexes that comprise gp96. 27.The method of claim 24, wherein steps (a) and (b) are repeated for 1 to12 cycles.
 28. The method of claim 24, comprising administering thepurified heat shock protein preparation once a week for the first 4weeks.
 29. The method of claim 28, comprising administering the purifiedheat shock protein once every other week after the first 4 weeks ofadministration.
 30. The method of claim 24, wherein the purified heatshock protein preparation and the Bevacizumab are administered 2 to 4days apart, 4 to 6 days apart, 1 week apart, 1 to 2 weeks apart, or 2 to4 weeks apart.
 31. The method of claim 24, which further comprises thestep of administering to the subject radiation therapy.
 32. The methodof claim 24, comprising administering the purified heat shock proteinpreparation for a first period of time, followed by administering theBevacizumab for a second period of time, and repeating this sequentialadministration.
 33. The method of claim 24, wherein the Bevacizumab isadministered prior to an initial administration of the purified heatshock protein preparation.
 34. The method of claim 24, wherein theBevacizumab is administered concurrently with the administration of thepurified heat shock protein preparation.
 35. The method of claim 24,wherein the Bevacizumab is administered subsequent to an initialadministration of the purified heat shock protein preparation.
 36. Themethod of claim 24, wherein the Bevacizumab and the purified heat shockprotein preparation are cyclically administered.
 37. The method of claim24, wherein the purified heat shock protein preparation is autologous tothe subject.
 38. The method of claim 24, wherein the subject is human.39. The method of claim 38, wherein the purified heat shock proteinpreparation comprises heat shock protein-peptide complexes isolated fromcells of the cancer of the subject.
 40. The method of claim 24, whereinthe purified heat shock protein preparation comprises heat shockprotein-peptide complexes comprising heat shock proteins non-covalentlyattached to antigenic peptides.
 41. The method of claim 24, wherein thepurified heat shock protein preparation comprises heat shockprotein-peptide complexes isolated from cells of the cancer.
 42. Themethod of claim 24, wherein the antigenic peptide displays antigenicityof a tumor-specific antigen or a tumor-associated antigen of the cancerin the subject.
 43. The method of claim 24, wherein the cancer is arelapse form of cancer.
 44. The method of claim 24, wherein theBevacizumab is administered no more than 24 hours after the purifiedheat shock protein preparation.
 45. The method of claim 24, wherein thepurified heat shock protein preparation is administered intradermally.46. The method of claim 45, wherein the Bevacizumab is administeredintravenously.
 47. A method of treating a cancer in a subject in needthereof, the method comprising: administering to the subject a purifiedheat shock protein preparation comprising a heat shock protein-peptidecomplex comprising a heat shock protein covalently or noncovalentlyattached to an antigenic peptide, wherein the antigenic peptide displaysantigenicity of an antigen of the cancer, wherein the subject is acandidate for receiving Bevacizumab to treat the cancer and wherein thesubject is administered Bevacizumab within 2 weeks or one month frombeing administered the purified heat shock protein preparation.
 48. Themethod of claim 47, wherein the Bevacizumab is administered subsequentto the administration of the purified heat shock protein preparation.49. The method of claim 47, wherein the Bevacizumab is administeredprior to the administration of the purified heat shock proteinpreparation.
 50. The method of claim 48, wherein the cancer is a relapseform of cancer.
 51. The method of claim 49, wherein the cancer is arelapse form of cancer.
 52. The method of claim 47, wherein the purifiedheat shock protein preparation comprises heat shock protein-peptidecomplexes that comprise gp96.
 53. The method of claim 47, wherein thepurified heat shock protein preparation is autologous to the subject.54. The method of claim 47, wherein the subject is human.
 55. The methodof claim 47, wherein the purified heat shock protein preparationcomprises heat shock protein-peptide complexes comprising heat shockproteins non-covalently attached to antigenic peptides.
 56. The methodof claim 47, wherein the antigenic peptide displays antigenicity of atumor-specific antigen or a tumor-associated antigen of the cancer inthe subject.
 57. The method of claim 47, wherein the subject isadministered Bevacizumab no more than 24 hours after the purified heatshock protein preparation.
 58. The method of claim 47, wherein thepurified heat shock protein preparation is administered intradermally.59. The method of claim 58, wherein the Bevacizumab is administeredintravenously.